Methods of diagnosing colorectal cancer, compositions, and methods of screening for colorectal cancer modulators

ABSTRACT

Described herein are methods that can be used for diagnosis and prognosis of colorectal cancer. Also described herein are methods that can be used to screen candidate bioactive agents for the ability to modulate colorectal cancer. Additionally, methods and molecular targets (genes and their products) for therapeutic intervention in colorectal cancer are described.

This application is a continuing application of U.S. application Ser.No. 09/525,993, filed on Mar. 15, 2000, which is a continuation-in-partof U.S. application Ser. No. 09/493,444 filed Jan. 28, 2000.

FIELD OF THE INVENTION

The invention relates to the identification of expression profiles andthe nucleic acids involved in colorectal cancer, and to the use of suchexpression profiles and nucleic acids in diagnosis and prognosis ofcolorectal cancer. The invention further relates to methods foridentifying and using candidate agents and/or targets which modulatecolorectal cancer.

BACKGROUND OF THE INVENTION

Colorectal cancer is a significant cancer in Western populations. Itdevelops as the result of a pathologic transformation of normal colonepithelium to an invasive cancer. There have been a number of recentlycharacterized genetic alterations that have been implicated incolorectal cancer, including mutations in two classes of genes,tumor-suppressor genes and proto-oncogenes, with recent work suggestingthat mutations in DNA repair genes may also be involved intumorigenesis. For example, inactivating mutations of both alleles ofthe adenomatous polyposis coli (APC) gene, a tumor suppressor gene,appears to be one of the earliest events in colorectal cancer, and mayeven be the initiating event. Other genes implicated in colorectalcancer include the MCC gene, the p53 gene, the DCC (deleted incolorectal carcinoma) gene and other chromosome 18q genes, and genes inthe TGF-β signalling pathway. For a review, see Molecular Biology ofColorectal Cancer, pp238-299, in Curr. Probl. Cancer, September/October1997.

Imaging of colorectal cancer for diagnosis has been problematic andlimited. In addition, dissemination of tumor cells (metastases) tolocoregional lymph nodes is an important prognostic factor; five yearsurvival rates drop from 80 percent in patients with no lymph nodemetastases to 45 to 50 percent in those patients who do have lymph nodemetastases. A recent report showed that micrometastases can be detectedfrom lymph nodes using reverse transcriptase-PCR methods based on thepresence of mRNA for carcinoembryonic antigen, which has previously beenshown to be present in the vast majority of colorectal cancers but notin normal tissues. Liefers et al., New England J. of Med. 339(4):223(1998).

Thus, methods that can be used for diagnosis and prognosis of colorectalcancer would be desirable. While academia and industry has made aneffort to identify novel sequences, there has not been an equal effortexerted to identify the function of the novel sequences in diseasestates of concern, such as cancer. For example, databases show thesequence for accession numbers AA411502 and AF216312, and the later hasbeen identified as a type II membrane serine protease, but there is nodata correlating these sequences with a disease state. Further providedare methods that can be used to screen candidate bioactive agents forthe ability to modulate colorectal cancer. Additionally, provided hereinare molecular targets for therapeutic intervention in colorectal andother cancers.

SUMMARY OF THE INVENTION

The present invention provides methods for screening for compositionswhich modulate colorectal cancer. In one aspect, a method of screeningdrug candidates comprises providing a cell that expresses an expressionprofile gene or fragments thereof. Preferred embodiments of theexpression profile gene as described herein include the sequencecomprising CJA8 or a fragment thereof. The method further includesadding a drug candidate to the cell and determining the effect of thedrug candidate on the expression of the expression profile gene.

In one embodiment, the method of screening drug candidates includescomparing the level of expression in the absence of the drug candidateto the level of expression in the presence of the drug candidate,wherein the concentration of the drug candidate can vary when present,and wherein the comparison can occur after addition or removal of thedrug candidate. In a preferred embodiment, the cell expresses at leasttwo expression profile genes. The profile genes may show an increase ordecrease.

Also provided herein is a method of screening for a bioactive agentcapable of binding to a colorectal cancer modulating protein (BCMP) or afragment thereof, the method comprising combining the BCMP or fragmentthereof and a candidate bioactive agent, and determining the binding ofthe candidate agent to the BCMP or fragment thereof. In a preferredembodiment, the BCMP is CJA8.

Further provided herein is a method for screening for a bioactive agentcapable of modulating the bioactivity of a BCMP or a fragment thereof.In one embodiment, the method comprises combining the BCMP or fragmentthereof and a candidate bioactive agent, and determining the effect ofthe candidate agent on the bioactivity of te BCMP or the fragmentthereof. In a preferred embodiment, the BCMP is CJA8.

Also provided herein is a method of evaluating the effect of a candidatecolorectal cancer drug comprising administering the drug to a transgenicanimal expressing or over-expressing a BCMP or a fragment thereof, or ananimal lacking a BCMP for example as a result of a gene knockout. In apreferred embodiment, the BCMP is CJA8.

Additionally, provided herein is a method of evaluating the effect of acandidate colorectal cancer drug comprising administering the drug to apatient and removing a cell sample from the patient. The expressionprofile of the cell is then determined. This method may further comprisecomparing the expression profile to an expression profile of a healthyindividual.

Furthermore, a method of diagnosing colorectal cancer is provided. Themethod comprises determining the expression of a gene which encodes CJA8or a fragment thereof in a first tissue type of a first individual, andcomparing this to the expression of the gene from a second unaffectedindividual. A difference in the expression indicates that the firstindividual has colorectal cancer.

In another aspect, the present invention provides an antibody whichspecifically binds to CJA8, or a fragment thereof. Preferably theantibody is a monoclonal antibody. The antibody can be a fragment of anantibody such as a single stranded antibody as further described herein,or can be conjugated to another molecule. In one embodiment, theantibody is a humanized antibody.

In one embodiment a method for screening for a bioactive agent capableof interfering with the binding of CJA8 or a fragment thereof and anantibody which binds to said CJA8 or fragment thereof is provided. In apreferred embodiment, the method comprises combining CJA8 or a fragmentthereof, a candidate bioactive agent and an antibody which binds to saidCJA8 or fragment thereof. The method further includes determining thebinding of said CJA8 or fragment thereof and said antibody. Whereinthere is a change in binding, an agent is identified as an interferingagent. The interfering agent can be an agonist or an antagonist.Preferably, the antibody as well as the agent inhibits colorectalcancer.

In one aspect of the invention, a method for inhibiting the activity ofa colorectal cancer modulating protein are provided. The methodcomprises binding an inhibitor to the protein. In a preferredembodiment, the protein is CJA8.

In another aspect, the invention provides a method for neutralizing theeffect of a colorectal cancer modulating protein. The method comprisescontacting an agent specific for the protein with the protein in anamount sufficient to effect neutralization. In a preferred embodiment,the protein is CJA8.

In a further aspect, a method for treating or inhibiting colorectalcancer is provided. In one embodiment, the method comprisesadministering to a cell a composition comprising an antibody to CJA8 ora fragment thereof. In one embodiment, the antibody is conjugated to atherapeutic moiety. Such therapeutic moieties include a cytotoxic agentand a radioisotope. The method can be performed in vitro or in vivo,preferably in vivo to an individual. In a preferred embodiment themethod of inhibiting colorectal cancer is provided to an individual withsuch cancer.

As described herein, methods of treating or inhibiting colorectal cancercan be performed by administering an inhibitor of CJA8 activity to acell or individual. In one embodiment, a CJA8 inhibitor is an antisensemolecule to a nucleic acid encoding CJA8.

Moreover, provided herein is a biochip comprising a nucleic acid segmentwhich encodes CJA8, or a fragment thereof, wherein the biochip comprisesfewer than 1000 nucleic acid probes. Preferably at least two nucleicacid segments are included.

Also provided herein are methods of eliciting an immune response in anindividual. In one embodiment a method provided herein comprisesadministering to an individual a composition comprising CJA8 or afragment thereof. In another aspect, said composition comprises anucleic acid comprising a sequence encoding CJA8 or a fragment thereof.

Further provided herein are compositions capable of eliciting an immuneresponse in an individual. In one embodiment, a composition providedherein comprises CJA8 or a fragment thereof and a pharmaceuticallyacceptable carrier. In another embodiment, said composition comprises anucleic acid comprising a sequence encoding CJA8 or a fragment thereofand a pharmaceutically acceptable carrier.

Other aspects of the invention will become apparent to the skilledartisan by the following description of the invention.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 (SEQ ID NO:1) shows an embodiment of a nucleic acid (mRNA) whichincludes a sequence which encodes a colorectal cancer protein providedherein, CJA8. The start and stop codons are shaded.

FIG. 2 (SEQ ID NO:2) shows an embodiment of an amino acid sequence ofCJA8. A putative transmembrane region is shaded.

FIG. 3 shows the relative amount of expression of CJA8 in varioussamples of colorectal cancer tissue(dark bars) and many normal tissuetypes (light bars).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel methods for diagnosis and prognosisevaluation for colorectal cancer, as well as methods for screening forcompositions which modulate colorectal cancer and compositions whichbind to modulators of colorectal cancer. In one aspect, the expressionlevels of genes are determined in different patient samples for whicheither diagnosis or prognosis information is desired, to provideexpression profiles. An expression profile of a particular sample isessentially a “fingerprint” of the state of the sample; while two statesmay have any particular gene similarly expressed, the evaluation of anumber of genes simultaneously allows the generation of a geneexpression profile that is unique to the state of the cell. That is,normal tissue may be distinguished from colorectal cancer tissue, andwithin colorectal cancer tissue, different prognosis states (good orpoor long term survival prospects, for example) may be determined. Bycomparing expression profiles of colorectal cancer tissue in differentstates, information regarding which genes are important (including bothup- and down-regulation of genes) in each of these states is obtained.The identification of sequences that are differentially expressed incolorectal cancer tissue versus normal colon tissue, as well asdifferential expression resulting in different prognostic outcomes,allows the use of this information in a number of ways. For example, theevaluation of a particular treatment regime may be evaluated: does achemotherapeutic drug act to improve the long-term prognosis in aparticular patient. Similarly, diagnosis may be done or confirmed bycomparing patient samples with the known expression profiles.Furthermore, these gene expression profiles (or individual genes) allowscreening of drug candidates with an eye to mimicking or altering aparticular expression profile; for example, screening can be done fordrugs that suppress the colorectal cancer expression profile or converta poor prognosis profile to a better prognosis profile. This may be doneby making biochips comprising sets of the important colorectal cancergenes, which can then be used in these screens. These methods can alsobe done on the protein basis; that is, protein expression levels of thecolorectal cancer proteins can be evaluated for diagnostic andprognostic purposes or to screen candidate agents. In addition, thecolorectal cancer nucleic acid sequences can be administered for genetherapy purposes, including the administration of antisense nucleicacids, or the colorectal cancer proteins (including antibodies and othermodulators thereof) administered as therapeutic drugs.

Thus the present invention provides nucleic acid and protein sequencesthat are differentially expressed in colorectal cancer when compared tonormal tissue. The differentially expressed sequences provided hereinare termed “colorectal cancer sequences”. As outlined below, colorectalcancer sequences include those that are up-regulated (i.e. expressed ata higher level) in colorectal cancer, as well as those that aredown-regulated (i.e. expressed at a lower level) in colorectal cancer.In a preferred embodiment, the colorectal cancer sequences are fromhumans; however, as will be appreciated by those in the art, colorectalcancer sequences from other organisms may be useful in animal models ofdisease and drug evaluation; thus, other colorectal cancer sequences areprovided, from vertebrates, including mammals, including rodents (rats,mice, hamsters, guinea pigs, etc.), primates, farm animals (includingsheep, goats, pigs, cows, horses, etc). Colorectal cancer sequences fromother organisms may be obtained using the techniques outlined below.

In a preferred embodiment, the colorectal cancer sequences are those ofnucleic acids encoding CJA8 or fragments thereof. Preferably, thecolorectal cancer sequences are those depicted in FIG. 1 (SEQ ID NO: 1),or fragments thereof. Preferably, the colorectal cancer sequences encodea protein having the amino acid sequence depicted in FIG. 2 (SEQ IDNO:2), or a fragment thereof. In a preferred embodiment, CJA8 has thesequence of a human type II membrane serine protease.

Colorectal cancer sequences can include both nucleic acid and amino acidsequences. In a preferred embodiment, the colorectal cancer sequencesare recombinant nucleic acids. By the term “recombinant nucleic acid”herein is meant nucleic acid, originally formed in vitro, in general, bythe manipulation of nucleic acid by polymerases and endonucleases, in aform not normally found in nature. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e. using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdepicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which itis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesat least about 75% by weight of the total protein, with at least about80% being preferred, and at least about 90% being particularlypreferred. The definition includes the production of a colorectal cancerprotein from one organism in a different organism or host cell.Alternatively, the protein may be made at a significantly higherconcentration than is normally seen, through the use of an induciblepromoter or high expression promoter, such that the protein is made atincreased concentration levels. Alternatively, the protein may be in aform not normally found in nature, as in the addition of an epitope tagor amino acid substitutions, insertions and deletions, as discussedbelow.

In a preferred embodiment, the colorectal cancer sequences are nucleicacids. As will be appreciated by those in the art and is more fullyoutlined below, colorectal cancer sequences are useful in a variety ofapplications, including diagnostic applications, which will detectnaturally occurring nucleic acids, as well as screening applications;for example, biochips comprising nucleic acid probes to the colorectalcancer sequences can be generated. In the broadest sense, then, by“nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within one definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News June 2,1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done for a varietyof reasons, for example to increase the stability and half-life of suchmolecules in physiological environments or as probes on a biochip.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C.Similarly, due to their non-ionic nature, hybridization of the basesattached to these backbones is relatively insensitive to saltconcentration. In addition, PNAs are not degraded by cellular enzymes,and thus can be more stable.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. As will be appreciated by those in the art, thedepiction of a single strand (“Watson”) also defines the sequence of theother strand (“Crick”); thus the sequences described herein alsoincludes the complement of the sequence. The nucleic acid may be DNA,both genomic and cDNA, RNA or a hybrid, where the nucleic acid containsany combination of deoxyribo- and ribo-nucleotides, and any combinationof bases, including uracil, adenine, thymine, cytosine, guanine,inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. As usedherein, the term “nucleoside” includes nucleotides and nucleoside andnucleotide analogs, and modified nucleosides such as amino modifiednucleosides. In addition, “nucleoside” includes non-naturally occurringanalog structures. Thus for example the individual units of a peptidenucleic acid, each containing a base, are referred to herein as anucleoside.

A colorectal cancer sequence can be initially identified by substantialnucleic acid and/or amino acid sequence homology to the colorectalcancer sequences outlined herein. Such homology can be based upon theoverall nucleic acid or amino acid sequence, and is generally determinedas outlined below, using either homology programs or hybridizationconditions.

The colorectal cancer sequences of the invention can be identified asfollows. Samples of normal and tumor tissue are applied to biochipscomprising nucleic acid probes. The samples are first microdissected, ifapplicable, and treated as is know in the art for the preparation ofmRNA. Suitable biochips are commercially available, for example fromAffymetrix. Gene expression profiles as described herein are generated,and the data analyzed.

In a preferred embodiment, the genes showing changes in expression asbetween normal and disease states are compared to genes expressed inother normal tissues, including, but not limited to lung, heart, brain,liver, colorectal, kidney, muscle, prostate, small intestine, largeintestine, spleen, bone, and placenta. In a preferred embodiment, thosegenes identified during the colorectal cancer screen that are expressedin any significant amount in other tissues are removed from the profile,although in some embodiments, this is not necessary. That is, whenscreening for drugs, it is preferable that the target be diseasespecific, to minimize possible side effects.

In a preferred embodiment, colorectal cancer sequences are those thatare up-regulated in colorectal cancer; that is, the expression of thesegenes is higher in colorectal carcinoma as compared to normal colontissue. “Up-regulation” as used herein means at least about a 50%increase, preferably a two-fold change, more preferably at least about athree fold change, with at least about five-fold or higher beingpreferred. All accession numbers herein are for the GenBank sequencedatabase and the sequences of the accession numbers are hereby expresslyincorporated by reference. GenBank is known in the art, see, e.g.,Benson, D A, et al., Nucleic Acids Research 26:1-7 (1998) andhftp://www.ncbi.nim.nih.gov/. In addition, these genes are found to beexpressed in a limited amount or not at all in bladder, bone marrow,brain, breast, fibroblasts, heart, kidney, liver, lung, muscle,pancreas, prostate, skin, small intestine, spleen, stomach and testes.

In a preferred embodiment, the gene for CJA8 is upregulated in coloncancer tissue as compared with normal colon tissue.

In another embodiment, colorectal cancer sequences are those that aredown-regulated in colorectal cancer; that is, the expression of thesegenes is lower in, for example, colorectal carcinoma as compared tonormal colon tissue. “Down-regulation” as used herein means at leastabout a two-fold change, preferably at least about a three fold change,with at least about five-fold or higher being preferred.

Colorectal cancer proteins of the present invention may be classified assecreted proteins, transmembrane proteins or intracellular proteins. Ina preferred embodiment the colorectal cancer protein is an intracellularprotein. Intracellular proteins may be found in the cytoplasm and/or inthe nucleus. Intracellular proteins are involved in all aspects ofcellular function and replication (including, for example, signalingpathways); aberrant expression of such proteins results in unregulatedor disregulated cellular processes. For example, many intracellularproteins have enzymatic activity such as protein kinase activity,protein phosphatase activity, protease activity, nucleotide cyclaseactivity, polymerase activity and the like. Intracellular proteins alsoserve as docking proteins that are involved in organizing complexes ofproteins, or targeting proteins to various subcellular localizations,and are involved in maintaining the structural integrity of organelles.

An increasingly appreciated concept in characterizing intracellularproteins is the presence in the proteins of one or more motifs for whichdefined functions have been attributed. In addition to the highlyconserved sequences found in the enzymatic domain of proteins, highlyconserved sequences have been identified in proteins that are involvedin protein-protein interaction. For example, Src-homology-2 (SH2)domains bind tyrosine-phosphorylated targets in a sequence dependentmanner. PTB domains, which are distinct from SH2 domains, also bindtyrosine phosphorylated targets. SH3 domains bind to proline-richtargets. In addition, PH domains, tetratricopeptide repeats and WDdomains to name only a few, have been shown to mediate protein-proteininteractions. Some of these may also be involved in binding tophospholipids or other second messengers. As will be appreciated by oneof ordinary skill in the art, these motifs can be identified on thebasis of primary sequence; thus, an analysis of the sequence of proteinsmay provide insight into both the enzymatic potential of the moleculeand/or molecules with which the protein may associate.

In a preferred embodiment, the colorectal cancer sequences aretransmembrane proteins. Transmembrane proteins are molecules that spanthe phospholipid bilayer of a cell. They may have an intracellulardomain, an extracellular domain, or both. The intracellular domains ofsuch proteins may have a number of functions including those alreadydescribed for intracellular proteins. For example, the intracellulardomain may have enzymatic activity and/or may serve as a binding sitefor additional proteins. Frequently the intracellular domain oftransmembrane proteins serves both roles. For example certain receptortyrosine kinases have both protein kinase activity and SH2 domains. Inaddition, autophosphorylation of tyrosines on the receptor moleculeitself, creates binding sites for additional SH2 domain containingproteins.

Transmembrane proteins may contain from one to many transmembranedomains. For example, receptor tyrosine kinases, certain cytokinereceptors, receptor guanylyl cyclases and receptor serine/threonineprotein kinases contain a single transmembrane domain. However, variousother proteins including channels and adenylyl cyclases contain numeroustransmembrane domains. Many important cell surface receptors areclassified as “seven transmembrane domain” proteins, as they contain 7membrane spanning regions. Important transmembrane protein receptorsinclude, but are not limited to insulin receptor, insulin-like growthfactor receptor, human growth hormone receptor, glucose transporters,transferrin receptor, epidermal growth factor receptor, low densitylipoprotein receptor, epidermal growth factor receptor, leptin receptor,interleukin receptors, e.g. IL-1 receptor, IL-2 receptor, etc.

Characteristics of transmembrane domains include approximately 20consecutive hydrophobic amino acids that may be followed by chargedamino acids. Therefore, upon analysis of the amino acid sequence of aparticular protein, the localization and number of transmembrane domainswithin the protein may be predicted.

The extracellular domains of transmembrane proteins are diverse;however, conserved motifs are found repeatedly among variousextracellular domains. Conserved structure and/or functions have beenascribed to different extracellular motifs. For example, cytokinereceptors are characterized by a cluster of cysteines and a WSXWS(W=tryptophan, S=serine, X=any amino acid; SEQ ID NO:3) motif.Immunoglobulin-like domains are highly conserved. Mucin-like domains maybe involved in cell adhesion and leucine-rich repeats participate inprotein-protein interactions.

Many extracellular domains are involved in binding to other molecules.In one aspect, extracellular domains are receptors. Factors that bindthe receptor domain include circulating ligands, which may be peptides,proteins, or small molecules such as adenosine and the like. Forexample, growth factors such as EGF, FGF and PDGF are circulating growthfactors that bind to their cognate receptors to initiate a variety ofcellular responses. Other factors include cytokines, mitogenic factors,neurotrophic factors and the like. Extracellular domains also bind tocell-associated molecules. In this respect, they mediate cell-cellinteractions. Cell-associated ligands can be tethered to the cell forexample via a glycosylphosphatidylinositol (GPI) anchor, or maythemselves be transmembrane proteins. Extracellular domains alsoassociate with the extracellular matrix and contribute to themaintenance of the cell structure.

Colorectal cancer proteins that are transmembrane are particularlypreferred in the present invention as they are good targets forimmunotherapeutics, as are described herein. In addition, as outlinedbelow, transmembrane proteins can be also useful in imaging modalities.

It will also be appreciated by those in the art that a transmembraneprotein can be made soluble by removing transmembrane sequences, forexample through recombinant methods. Furthermore, transmembrane proteinsthat have been made soluble can be made to be secreted throughrecombinant means by adding an appropriate signal sequence.

In a preferred embodiment, the colorectal cancer proteins are secretedproteins; the secretion of which can be either constitutive orregulated. These proteins have a signal peptide or signal sequence thattargets the molecule to the secretory pathway. Secreted proteins areinvolved in numerous physiological events; by virtue of theircirculating nature, they serve to transmit signals to various other celltypes. The secreted protein may function in an autocrine manner (actingon the cell that secreted the factor), a paracrine manner (acting oncells in close proximity to the cell that secreted the factor) or anendocrine manner (acting on cells at a distance). Thus secretedmolecules find use in modulating or altering numerous aspects ofphysiology. Colorectal cancer proteins that are secreted proteins areparticularly preferred in the present invention as they serve as goodtargets for diagnostic markers, for example for blood tests.

A colorectal cancer sequence is initially identified by substantialnucleic acid and/or amino acid sequence homology to the colorectalcancer sequences outlined herein. Such homology can be based upon theoverall nucleic acid or amino acid sequence, and is generally determinedas outlined below, using either homology programs or hybridizationconditions.

As used herein, a nucleic acid is a “colorectal cancer nucleic acid” ifthe overall homology of the nucleic acid sequence to the nucleic acidsequences encoding the amino acid sequences of the figures is preferablygreater than about 75%, more preferably greater than about 80%, evenmore preferably greater than about 85% and most preferably greater than90%. In some embodiments the homology will be as high as about 93 to 95or 98%. Homology in this context means sequence similarity or identity,with identity being preferred. A preferred comparison for homologypurposes is to compare the sequence containing sequencing errors to thecorrect sequence. This homology will be determined using standardtechniques known in the art, including, but not limited to, the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),by the homology alignment algorithm of Needleman & Wunsch, J. Mol.Biool. 48:443 (1970), by the search for similarity method of Pearson &Lipman, PNAS USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12:387-395 (1984), preferably using the defaultsettings, or by inspection.

In a preferred embodiment, the sequences which are used to determinesequence identity or similarity are selected from the sequences setforth in the figures, preferably those shown in FIG. 1 (SEQ ID NO:1) andfragments thereof. In one embodiment the sequences utilized herein arethose set forth in the figures. In another embodiment, the sequences arenaturally occurring allelic variants of the sequence set forth in thefigures. In another embodiment, the sequences are sequenced variants asfurther described herein.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266: 460-480 (1996)[http://blast.wustl/edu/blast/ READ.html]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. A % amino acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “longer” sequence in the aligned region.The “longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-Blast-2 to maximize the alignmentscore are ignored).

Thus, “percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotide residues in a candidate sequence that areidentical with the nucleotide residues of FIG. 1 (SEQ ID NO:1). Apreferred method utilizes the BLASTN module of WU-BLAST-2 set to thedefault parameters, with overlap span and overlap fraction set to 1 and0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer nucleosides than those of the figures, it is understood that thepercentage of homology will be determined based on the number ofhomologous nucleosides in relation to the total number of nucleosides.Thus, for example, homology of sequences shorter than those of thesequences identified herein and as discussed below, will be determinedusing the number of nucleosides in the shorter sequence.

In one embodiment, the nucleic acid homology is determined throughhybridization studies. Thus, for example, nucleic acids which hybridizeunder high stringency to the nucleic acid sequences which encode thepeptides identified in the figures, or their complements, are considereda colorectal cancer sequence. High stringency conditions are known inthe art; see for example Maniatis et al., Molecular Cloning: ALaboratory Manual, 2d Edition, 1989, and Short Protocols in MolecularBiology, ed. Ausubel, et al., both of which are hereby incorporated byreference. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, “Overview of principles of hybridization and the strategy ofnucleic acid assays” (1993). Generally, stringent conditions areselected to be about 5-10° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g. 10 to 50nucleotides) and at least about 60° C. for long probes (e.g. greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

In addition, the colorectal cancer nucleic acid sequences of theinvention are fragments of larger genes, i.e. they are nucleic acidsegments. “Genes” in this context includes coding regions, non-codingregions, and mixtures of coding and non-coding regions. Accordingly, aswill be appreciated by those in the art, using the sequences providedherein, additional sequences of the colorectal cancer genes can beobtained, using techniques well known in the art for cloning eitherlonger sequences or the full length sequences; see Maniatis et al., andAusubel, et al., supra, hereby expressly incorporated by reference.

Once the colorectal cancer nucleic acid is identified, it can be clonedand, if necessary, its constituent parts recombined to form the entirecolorectal cancer nucleic acid. Once isolated from its natural source,e.g., contained within a plasmid or other vector or excised therefrom asa linear nucleic acid segment, the recombinant colorectal cancer nucleicacid can be further-used as a probe to identify and isolate othercolorectal cancer nucleic acids, for example additional coding regions.It can also be used as a “precursor” nucleic acid to make modified orvariant colorectal cancer nucleic acids and proteins.

The colorectal cancer nucleic acids of the present invention are used inseveral ways. In a first embodiment, nucleic acid probes to thecolorectal cancer nucleic acids are made and attached to biochips to beused in screening and diagnostic methods, as outlined below, or foradministration, for example for gene therapy and/or antisenseapplications. Alternatively, the colorectal cancer nucleic acids thatinclude coding regions of colorectal cancer proteins can be put intoexpression vectors for the expression of colorectal cancer proteins,again either for screening purposes or for administration to a patient.

In a preferred embodiment, nucleic acid probes to colorectal cancernucleic acids (both the nucleic acid sequences encoding peptidesoutlined in the figures and/or the complements thereof) are made. Thenucleic acid probes attached to the biochip are designed to besubstantially complementary to the colorectal cancer nucleic acids, i.e.the target sequence (either the target sequence of the sample or toother probe sequences, for example in sandwich assays), such thathybridization of the target sequence and the probes of the presentinvention occurs. As outlined below, this complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions,particularly high stringency conditions, as outlined herein.

A nucleic acid probe is generally single stranded but can be partiallysingle and partially double stranded. The strandedness of the probe isdictated by the structure, composition, and properties of the targetsequence. In general, the nucleic acid probes range from about 8 toabout 100 bases long, with from about 10 to about 80 bases beingpreferred, and from about 30 to about 50 bases being particularlypreferred. That is, generally whole genes are not used. In someembodiments, much longer nucleic acids can be used, up to hundreds ofbases.

In a preferred embodiment, more than one probe per sequence is used,with either overlapping probes or probes to different sections of thetarget being used. That is, two, three, four or more probes, with threebeing preferred, are used to build in a redundancy for a particulartarget. The probes can be overlapping (i.e. have some sequence incommon), or separate.

As will be appreciated by those in the art, nucleic acids can beattached or immobilized to a solid support in a wide variety of ways. By“immobilized” and grammatical equivalents herein is meant theassociation or binding between the nucleic acid probe and the solidsupport is sufficient to be stable under the conditions of binding,washing, analysis, and removal as outlined below. The binding can becovalent or non-covalent. By “non-covalent binding” and grammaticalequivalents herein is meant one or more of either electrostatic,hydrophilic, and hydrophobic interactions. Included in non-covalentbinding is the covalent attachment of a molecule, such as, streptavidinto the support and the non-covalent binding of the biotinylated probe tothe streptavidin. By “covalent binding” and grammatical equivalentsherein is meant that the two moieties, the solid support and the probe,are attached by at least one bond, including sigma bonds, pi bonds andcoordination bonds. Covalent bonds can be formed directly between theprobe and the solid support or can be formed by a cross linker or byinclusion of a specific reactive group on either the solid support orthe probe or both molecules. Immobilization may also involve acombination of covalent and non-covalent interactions.

In general, the probes are attached to the biochip in a wide variety ofways, as will be appreciated by those in the art. As described herein,the nucleic acids can either be synthesized first, with subsequentattachment to the biochip, or can be directly synthesized on thebiochip.

The biochip comprises a suitable solid substrate. By “substrate” or“solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of the nucleic acid probesand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates are very large,and include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, etc. In general, the substrates allow optical detection and donot appreciably fluorescese. A preferred substrate is described incopending application entitled Reusable Low Fluorescent Plastic Biochipfiled Mar. 15, 1999, herein incorporated by reference in its entirety.

Generally the substrate is planar, although as will be appreciated bythose in the art, other configurations of substrates may be used aswell. For example, the probes may be placed on the inside surface of atube, for flow-through sample analysis to minimize sample volume.Similarly, the substrate may be flexible, such as a flexible foam,including closed cell foams made of particular plastics.

In a preferred embodiment, the surface of the biochip and the probe maybe derivatized with chemical functional groups for subsequent attachmentof the two. Thus, for example, the biochip is derivatized with achemical functional group including, but not limited to, amino groups,carboxy groups, oxo groups and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the probes can beattached using functional groups on the probes. For example, nucleicacids containing amino groups can be attached to surfaces comprisingamino groups, for example using linkers as are known in the art; forexample, homo-or hetero-bifunctional linkers as are well known (see 1994Pierce Chemical Company catalog, technical section on cross-linkers,pages 155-200, incorporated herein by reference). In addition, in somecases, additional linkers, such as alkyl groups (including substitutedand heteroalkyl groups) may be used.

In this embodiment, the oligonucleotides are synthesized as is known inthe art, and then attached to the surface of the solid support. As willbe appreciated by those skilled in the art, either the 5′ or 3′ terminusmay be attached to the solid support, or attachment may be via aninternal nucleoside.

In an additional embodiment, the immobilization to the solid support maybe very strong, yet non-covalent. For example, biotinylatedoligonucleotides can be made, which bind to surfaces covalently coatedwith streptavidin, resulting in attachment.

Alternatively, the oligonucleotides may be synthesized on the surface,as is known in the art. For example, photoactivation techniquesutilizing photopolymerization compounds and techniques are used. In apreferred embodiment, the nucleic acids can be synthesized in situ,using well known photolithographic techniques, such as those describedin WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; andreferences cited within, all of which are expressly incorporated byreference; these methods of attachment form the basis of the AffimetrixGeneChip™ technology.

In a preferred embodiment, colorectal cancer nucleic acids encodingcolorectal cancer proteins are used to make a variety of expressionvectors to express colorectal cancer proteins which can then be used inscreening assays, as described below. The expression vectors may beeither self-replicating extrachromosomal vectors or vectors whichintegrate into a host genome. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the colorectal cancerprotein. The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the colorectal cancer protein; for example,transcriptional and translational regulatory nucleic acid sequences fromBacillus are preferably used to express the colorectal cancer protein inBacillus. Numerous types of appropriate expression vectors, and suitableregulatory sequences are known in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

The colorectal cancer proteins of the present invention are produced byculturing a host cell transformed with an expression vector containingnucleic acid encoding a colorectal cancer protein, under the appropriateconditions to induce or cause expression of the colorectal cancerprotein. The conditions appropriate for colorectal cancer proteinexpression will vary with the choice of the expression vector and thehost cell, and will be easily ascertained by one skilled in the artthrough routine experimentation. For example, the use of constitutivepromoters in the expression vector will require optimizing the growthand proliferation of the host cell, while the use of an induciblepromoter requires the appropriate growth conditions for induction. Inaddition, in some embodiments, the timing of the harvest is important.For example, the baculoviral systems used in insect cell expression arelytic viruses, and thus harvest time selection can be crucial forproduct yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melangaster cells, Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293cells, Neurospora, BHK, CHO, COS, HeLa cells, THP1 cell line (amacrophage cell line) and human cells and cell lines.

In a preferred embodiment, the colorectal cancer proteins are expressedin mammalian cells. Mammalian expression systems are also known in theart, and include retroviral systems. A preferred expression vectorsystem is a retroviral vector system such as is generally described inPCT/US97/01019 and PCT/US97/01048, both of which are hereby expresslyincorporated by reference. Of particular use as mammalian promoters arethe promoters from mammalian viral genes, since the viral genes areoften highly expressed and have a broad host range. Examples include theSV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirusmajor late promoter, herpes simplex virus promoter, and the CMVpromoter. Typically, transcription termination and polyadenylationsequences recognized by mammalian cells are regulatory regions located3′ to the translation stop codon and thus, together with the promoterelements, flank the coding sequence. Examples of transcriptionterminator and polyadenlytion signals include those derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, colorectal cancer proteins are expressed inbacterial systems. Bacterial expression systems are well known in theart. Promoters from bacteriophage may also be used and are known in theart. In addition, synthetic promoters and hybrid promoters are alsouseful; for example, the tac promoter is a hybrid of the trp and lacpromoter sequences. Furthermore, a bacterial promoter can includenaturally occurring promoters of non-bacterial origin that have theability to bind bacterial RNA polymerase and initiate transcription. Inaddition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. The expression vector may also include asignal peptide sequence that provides for secretion of the colorectalcancer protein in bacteria. The protein is either secreted into thegrowth media (gram-positive bacteria) or into the periplasmic space,located between the inner and outer membrane of the cell (gram-negativebacteria). The bacterial expression vector may also include a selectablemarker gene to allow for the selection of bacterial strains that havebeen transformed. Suitable selection genes include genes which renderthe bacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways. These components areassembled into expression vectors.

Expression vectors for bacteria are well known in the art, and includevectors for Bacillus subtilis, E. coli, Streptococcus cremoris, andStreptococcus lividans, among others. The bacterial expression vectorsare transformed into bacterial host cells using techniques well known inthe art, such as calcium chloride treatment, electroporation, andothers.

In one embodiment, colorectal cancer proteins are produced in insectcells. Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

In a preferred embodiment, colorectal cancer protein is produced inyeast cells. Yeast expression systems are well known in the art, andinclude expression vectors for Saccharomyces cerevisiae, Candidaalbicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilisand K. lactis, Pichia guillerimondii and P. pastoris,Schizosaccharomyces pombe, and Yarrowia lipolytica.

The colorectal cancer protein may also be made as a fusion protein,using techniques well known in the art. Thus, for example, for thecreation of monoclonal antibodies, if the desired epitope is small, thecolorectal cancer protein may be fused to a carrier protein to form animmunogen. Alternatively, the colorectal cancer protein may be made as afusion protein to increase expression, or for other reasons. Forexample, when the colorectal cancer protein is a colorectal cancerpeptide, the nucleic acid encoding the peptide may be linked to othernucleic acid for expression purposes.

In one embodiment, the colorectal cancer nucleic acids, proteins andantibodies of the invention are labeled. By “labeled” herein is meantthat a compound has at least one element, isotope or chemical compoundattached to enable the detection of the compound. In general, labelsfall into three classes: a) isotopic labels, which may be radioactive orheavy isotopes; b) immune labels, which may be antibodies or antigens;and c) colored or fluorescent dyes. The labels may be incorporated intothe colorectal cancer nucleic acids, proteins and antibodies at anyposition. For example, the label should be capable of producing, eitherdirectly or indirectly, a detectable signal. The detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compoun as fluorescein isothiocyanate, rhodamine, orluciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the label may be employed, includingthose methods described by Hunter et al., Nature, 144:945 (1962); Davidet al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,40:219 (1981); and Nygren, J. Histochem, and Cytochem., 30:407 (1982).

Accordingly, the present invention also provides colorectal cancerprotein sequences. A colorectal cancer protein of the present inventionmay be identified in several ways. “Protein” in this sense includesproteins, polypeptides, and peptides. As will be appreciated by those inthe art, the nucleic acid sequences of the invention can be used togenerate protein sequences. There are a variety of ways to do this,including cloning the entire gene and verifying its frame and amino acidsequence, or by comparing it to known sequences to search for homologyto provide a frame, assuming the colorectal cancer protein has homologyto some protein in the database being used. Generally, the nucleic acidsequences are input into a program that will search all three frames forhomology. This is done in a preferred embodiment using the followingNCBI Advanced BLAST parameters. The program is blastx or blastn. Thedatabase is nr. The input data is as “Sequence in FASTA format”. Theorganism list is “none”. The “expect” is 10; the filter is default. The“descriptions” is 500, the “alignments” is 500, and the “alignment view”is pairwise. The “Query Genetic Codes” is standard (1). The matrix isBLOSUM62; gap existence cost is 11, per residue gap cost is 1; and thelambda ratio is 0.85 default. This results in the generation of aputative protein sequence.

Also included within one embodiment of colorectal cancer proteins areamino acid variants of the naturally occurring sequences, as determinedherein. Preferably, the variants are preferably greater than about 75%homologous to the wild-type sequence, more preferably greater than about80%, even more preferably greater than about 85% and most preferablygreater than 90%. In some embodiments the homology will be as high asabout 93 to 95 or 98%. As for nucleic acids, homology in this contextmeans sequence similarity or identity, with identity being preferred.This homology will be determined using standard techniques known in theart as are outlined above for the nucleic acid homologies.

Colorectal cancer proteins of the present invention may be shorter orlonger than the wild type amino acid sequences. Thus, in a preferredembodiment, included within the definition of colorectal cancer proteinsare portions or fragments of the wild type sequences herein. Inaddition, as outlined above, the colorectal cancer nucleic acids of theinvention may be used to obtain additional coding regions, and thusadditional protein sequence, using techniques known in the art.

In a preferred embodiment, the colorectal cancer proteins are derivativeor variant colorectal cancer proteins as compared to the wild-typesequence. That is, as outlined more fully below, the derivativecolorectal cancer peptide will contain at least one amino acidsubstitution, deletion or insertion, with amino acid substitutions beingparticularly preferred. The amino acid substitution, insertion ordeletion may occur at any residue within the colorectal cancer peptide.

Also included in an embodiment of colorectal cancer proteins of thepresent invention are amino acid sequence variants. These variants fallinto one or more of three classes: substitutional, insertional ordeletional variants. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the colorectalcancer protein, using cassette or PCR mutagenesis or other techniqueswell known in the art, to produce DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture as outlinedabove. However, variant colorectal cancer protein fragments having up toabout 100-150 residues may be prepared by in vitro synthesis usingestablished techniques. Amino acid sequence variants are characterizedby the predetermined nature of the variation, a feature that sets themapart from naturally occurring allelic or interspecies variation of thecolorectal cancer protein amino acid sequence. The variants typicallyexhibit the same qualitative biological activity as the naturallyoccurring analogue, although variants can also be selected which havemodified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed colorectal cancer variantsscreened for the optimal combination of desired activity. Techniques formaking substitution mutations at predetermined sites in DNA having aknown sequence are well known, for example, M13 primer mutagenesis andPCR mutagenesis. Screening of the mutants is done using assays ofcolorectal cancer protein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the colorectal cancerprotein are desired, substitutions are generally made in accordance withthe following chart:

CHART 1 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart 1. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the colorectal cancer proteins as needed.Alternatively, the variant may be designed such that the biologicalactivity of the colorectal cancer protein is altered. For example,glycosylation sites may be altered or removed.

Covalent modifications of colorectal cancer polypeptides are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of a colorectal cancerpolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N-or C-terminal residues of acolorectal cancer polypeptide. Derivatization with bifunctional agentsis useful, for instance, for crosslinking colorectal cancer to awater-insoluble support matrix or surface for use in the method forpurifying anti-colorectal cancer antibodies or screening assays, as ismore fully described below. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl, threonyl or tyrosyl residues, methylation ofthe α-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the colorectal cancerpolypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencecolorectal cancer polypeptide, and/or adding one or more glycosylationsites that are not present in the native sequence colorectal cancerpolypeptide.

Addition of glycosylation sites to colorectal cancer polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence colorectalcancer polypeptide (for O-linked glycosylation sites). The colorectalcancer amino acid sequence may optionally be altered through changes atthe DNA level, particularly by mutating the DNA encoding the colorectalcancer polypeptide at preselected bases such that codons are generatedthat will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thecolorectal cancer polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,colorectal cancer Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the colorectal cancerpolypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo-andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of colorectal cancer proteincomprises linking the colorectal cancer polypeptide to one of a varietyof nonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Colorectal cancer polypeptides of the present invention may also bemodified in a way to form chimeric molecules comprising a colorectalcancer polypeptide fused to another, heterologous polypeptide or aminoacid sequence. In one embodiment, such a chimeric molecule comprises afusion of a colorectal cancer polypeptide with a tag polypeptide whichprovides an epitope to which an anti-tag antibody can selectively bind.The epitope tag is generally placed at the amino-or carboxyl-terminus ofthe colorectal cancer polypeptide. The presence of such epitope-taggedforms of a colorectal cancer polypeptide can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the colorectal cancer polypeptide to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of a colorectalcancer polypeptide with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3 7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610‘(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag andits antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

Also included with the definition of colorectal cancer protein in oneembodiment are other colorectal cancer proteins of the colorectal cancerfamily, and colorectal cancer proteins from other organisms, which arecloned and expressed as outlined below. Thus, probe or degeneratepolymerase chain reaction (PCR) primer sequences may be used to findother related colorectal cancer proteins from humans or other organisms.As will be appreciated by those in the art, particularly useful probeand/or PCR primer sequences include the unique areas of the colorectalcancer nucleic acid sequence. As is generally known in the art,preferred PCR primers are from about 15 to about 35 nucleotides inlength, with from about 20 to about 30 being preferred, and may containinosine as needed. The conditions for the PCR reaction are well known inthe art.

In addition, as is outlined herein, colorectal cancer proteins can bemade that are longer than those depicted in the figures, for example, bythe elucidation of additional sequences, the addition of epitope orpurification tags, the addition of other fusion sequences, etc.

Colorectal cancer proteins may also be identified as being encoded bycolorectal cancer nucleic acids.

Thus, colorectal cancer proteins are encoded by nucleic acids that willhybridize to the sequences of the sequence listings, or theircomplements, as outlined herein.

In a preferred embodiment, when the colorectal cancer protein is to beused to generate antibodies, for example for immunotherapy, thecolorectal cancer protein should share at least one epitope ordeterminant with the full length protein. By “epitope” or “determinant”herein is meant a portion of a protein which will generate and/or bindan antibody or T-cell receptor in the context of MHC. Thus, in mostinstances, antibodies made to a smaller colorectal cancer protein willbe able to bind to the full length protein. In a preferred embodiment,the epitope is unique; that is, antibodies generated to a unique epitopeshow little or no cross-reactivity.

In one embodiment, the term “antibody” includes antibody fragments, asare known in the art, including Fab, Fab₂, single chain antibodies (Fvfor example), chimeric antibodies, etc., either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies.

Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the CJA8 or fragmentthereof or a fusion protein thereof. It may be useful to conjugate theimmunizing agent to a protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include but arenot limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The antibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro. The immunizing agent will typically include the CJA8 polypeptideor fragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes (“PBLs”) are used if cells of human originare desired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

In one embodiment, the antibodies are bispecific antibodies. Bispecificantibodies are monoclonal, preferably human or humanized, antibodiesthat have binding specificities for at least two different antigens. Inthe present case, one of the binding specificities is for the CJA8 or afragment thereof, the other one is for any other antigen, and preferablyfor a cell-surface protein or receptor or receptor subunit, preferablyone that is tumor specific.

In a preferred embodiment, the antibodies to colorectal cancer arecapable of reducing or eliminating the biological function of colorectalcancer, as is described below. That is, the addition of anti-colorectalcancer antibodies (either polyclonal or preferably monoclonal) tocolorectal cancer (or cells containing colorectal cancer) may reduce oreliminate the colorectal cancer activity. Generally, at least a 25%decrease in activity is preferred, with at least about 50% beingparticularly preferred and about a 95-100% decrease being especiallypreferred.

In a preferred embodiment the antibodies to the colorectal cancerproteins are humanized antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric molecules of immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues form a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. OP. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

By immunotherapy is meant treatment of colorectal cancer with anantibody raised against colorectal cancer proteins. As used herein,immunotherapy can be passive or active. Passive immunotherapy as definedherein is the passive transfer of antibody to a recipient (patient).Active immunization is the induction of antibody and/or T-cell responsesin a recipient (patient). Induction of an immune response is the resultof providing the recipient with an antigen to which antibodies areraised. As appreciated by one of ordinary skill in the art, the antigenmay be provided by injecting a polypeptide against which antibodies aredesired to be raised into a recipient, or contacting the recipient witha nucleic acid capable of expressing the antigen and under conditionsfor expression of the antigen.

In a preferred embodiment the colorectal cancer proteins against whichantibodies are raised are secreted proteins as described above. Withoutbeing bound by theory, antibodies used for treatment, bind and preventthe secreted protein from binding to its receptor, thereby inactivatingthe secreted colorectal cancer protein.

In another preferred embodiment, the colorectal cancer protein to whichantibodies are raised is a transmembrane protein. Without being bound bytheory, antibodies used for treatment, bind the extracellular domain ofthe colorectal cancer protein and prevent it from binding to otherproteins, such as circulating ligands or cell-associated molecules. Theantibody may cause down-regulation of the transmembrane colorectalcancer protein. As will be appreciated by one of ordinary skill in theart, the antibody may be a competitive, non-competitive or uncompetitiveinhibitor of protein binding to the extracellular domain of thecolorectal cancer protein. The antibody is also an antagonist of thecolorectal cancer protein. Further, the antibody prevents activation ofthe transmembrane colorectal cancer protein. In one aspect, when theantibody prevents the binding of other molecules to the colorectalcancer protein, the antibody prevents growth of the cell. The antibodyalso sensitizes the cell to cytotoxic agents, including, but not limitedto TNF-α, TNF-β, IL-1, INF-γ and IL-2, or chemotherapeutic agentsincluding 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, andthe like. In some instances the antibody belongs to a sub-type thatactivates serum complement when complexed with the transmembrane proteinthereby mediating cytotoxicity. Thus, colorectal cancer is treated byadministering to a patient antibodies directed against the transmembranecolorectal cancer protein.

In another preferred embodiment, the antibody is conjugated to atherapeutic moiety. In one aspect the therapeutic moiety is a smallmolecule that modulates the activity of the colorectal cancer protein.In another aspect the therapeutic moiety modulates the activity ofmolecules associated with or in close proximity to the colorectal cancerprotein. The therapeutic moiety may inhibit enzymatic activity such asprotease or protein kinase activity associated with colorectal cancer.

In a preferred embodiment, the therapeutic moiety may also be acytotoxic agent. In this method, targeting the cytotoxic agent to tumortissue or cells, results in a reduction in the number of afflictedcells, thereby reducing symptoms associated with colorectal cancer.Cytotoxic agents are numerous and varied and include, but are notlimited to, cytotoxic drugs or toxins or active fragments of suchtoxins. Suitable toxins and their corresponding fragments includediptheria A chain, exotoxin A chain, ricin A chain, abrin A chain,curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents alsoinclude radiochemicals made by conjugating radioisotopes to antibodiesraised against colorectal cancer proteins, or binding of a radionuclideto a chelating agent that has been covalently attached to the antibody.Targeting the therapeutic moiety to transmembrane colorectal cancerproteins not only serves to increase the local concentration oftherapeutic moiety in the colorectal cancer afflicted area, but alsoserves to reduce deleterious side effects that may be associated withthe therapeutic moiety.

In another preferred embodiment, the PC protein against which theantibodies are raised is an intracellular protein. In this case, theantibody may be conjugated to a protein which facilitates entry into thecell. In one case, the antibody enters the cell by endocytosis. Inanother embodiment, a nucleic acid encoding the antibody is administeredto the individual or cell. Moreover, wherein the PC protein can betargeted within a cell, i.e., the nucleus, an antibody thereto containsa signal for that target localization, i.e., a nuclear localizationsignal.

The colorectal cancer antibodies of the invention specifically bind tocolorectal cancer proteins. By “specifically bind” herein is meant thatthe antibodies bind to the protein with a binding constant in the rangeof at least 10⁻⁴-10⁻⁶ M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹ M⁻¹.

In a preferred embodiment, the colorectal cancer protein is purified orisolated after expression. Colorectal cancer proteins may be isolated orpurified in a variety of ways known to those skilled in the artdepending on what other components are present in the sample. Standardpurification methods include electrophoretic, molecular, immunologicaland chromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.

For example, the colorectal cancer protein may be purified using astandard anti-colorectal cancer antibody column. Ultrafiltration anddiafiltration techniques, in conjunction with protein concentration, arealso useful. For general guidance in suitable purification techniques,see Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982). Thedegree of purification necessary will vary depending on the use of thecolorectal cancer protein. In some instances no purification will benecessary.

Once expressed and purified if necessary, the colorectal cancer proteinsand nucleic acids are useful in a number of applications.

In one aspect, the expression levels of genes are determined fordifferent cellular states in the colorectal cancer phenotype; that is,the expression levels of genes in normal colon tissue and in colorectalcancer tissue (and in some cases, for varying severities of colorectalcancer that relate to prognosis, as outlined below) are evaluated toprovide expression profiles. An expression profile of a particular cellstate or point of development is essentially a “fingerprint” of thestate; while two states may have any particular gene similarlyexpressed, the evaluation of a number of genes simultaneously allows thegeneration of a gene expression profile that is unique to the state ofthe cell. By comparing expression profiles of cells in different states,information regarding which genes are important (including both up- anddown-regulation of genes) in each of these states is obtained. Then,diagnosis may be done or confirmed: does tissue from a particularpatient have the gene expression profile of normal or colorectal cancertissue.

“Differential expression,” or grammatical equivalents as used herein,refers to both qualitative as well as quantitative differences in thegenes' temporal and/or cellular expression patterns within and among thecells. Thus, a colorectal cancer gene can qualitatively have itsexpression altered, including an activation or inactivation, in, forexample, normal versus colorectal cancer tissue. That is, genes may beturned on or turned off in a particular state, relative to anotherstate. As is apparent to the skilled artisan, any comparison of two ormore states can be made. Such a qualitatively regulated gene willexhibit an expression pattern within a state or cell type which isdetectable by standard techniques in one such state or cell type, but isnot detectable in both. Alternatively, the determination is quantitativein that expression is increased or decreased; that is, the expression ofthe gene is either upregulated, resulting in an increased amount oftranscript, or downregulated, resulting in a decreased amount oftranscript. The degree to which expression differs need only be largeenough to quantify via standard characterization techniques as outlinedbelow, such as by use of Affymetrix GeneChip™ expression arrays,Lockhart, Nature Biotechnology, 14:1675-1680 (1996), hereby expresslyincorporated by reference. Other techniques include, but are not limitedto, quantitative reverse transcriptase PCR, Northern analysis and RNaseprotection. As outlined above, preferably the change in expression (i.e.upregulation or downregulation) is at least about 50%, more preferablyat least about 100%, more preferably at least about 150%, morepreferably, at least about 200%, with from 300 to at least 1000% beingespecially preferred.

As will be appreciated by those in the art, this may be done byevaluation at either the gene transcript, or the protein level; that is,the amount of gene expression may be monitored using nucleic acid probesto the DNA or RNA equivalent of the gene transcript, and thequantification of gene expression levels, or, alternatively, the finalgene product itself (protein) can be monitored, for example through theuse of antibodies to the colorectal cancer protein and standardimmunoassays (ELISAs,etc.) or other techniques, including massspectroscopy assays, 2D gel electrophoresis assays, etc. Thus, theproteins corresponding to colorectal cancer genes, i.e. those identifiedas being important in a colorectal cancer phenotype, can be evaluated ina colorectal cancer diagnostic test.

In a preferred embodiment, gene expression monitoring is done and anumber of genes, i.e. an expression profile, is monitoredsimultaneously, although multiple protein expression monitoring can bedone as well. Similarly, these assays may be done on an individual basisas well.

In this embodiment, the colorectal cancer nucleic acid probes areattached to biochips as outlined herein for the detection andquantification of colorectal cancer sequences in a particular cell. Theassays are further described below in the example.

In a preferred embodiment nucleic acids encoding the colorectal cancerprotein are detected. Although DNA or RNA encoding the colorectal cancerprotein may be detected, of particular interest are methods wherein themRNA encoding a colorectal cancer protein is detected. The presence ofmRNA in a sample is an indication that the colorectal cancer gene hasbeen transcribed to form the mRNA, and suggests that the protein isexpressed. Probes to detect the mRNA can be anynucleotide/deoxynucleotide probe that is complementary to and base pairswith the mRNA and includes but is not limited to oligonucleotides, cDNAor RNA. Probes also should contain a detectable label, as definedherein. In one method the mRNA is detected after immobilizing thenucleic acid to be examined on a solid support such as nylon membranesand hybridizing the probe with the sample. Following washing to removethe non-specifically bound probe, the label is detected. In anothermethod detection of the mRNA is performed in situ. In this methodpermeabilized cells or tissue samples are contacted with a detectablylabeled nucleic acid probe for sufficient time to allow the probe tohybridize with the target mRNA. Following washing to remove thenon-specifically bound probe, the label is detected. For example adigoxygenin labeled riboprobe (RNA probe) that is complementary to themRNA encoding a colorectal cancer protein is detected by binding thedigoxygenin with an anti-digoxygenin secondary antibody and developedwith nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.

In a preferred embodiment, any of the three classes of proteins asdescribed herein (secreted, transmembrane or intracellular proteins) areused in diagnostic assays. The colorectal cancer proteins, antibodies,nucleic acids, modified proteins and cells containing colorectal cancersequences are used in diagnostic assays. This can be done on anindividual gene or corresponding polypeptide level. In a preferredembodiment, the expression profiles are used, preferably in conjunctionwith high throughput screening techniques to allow monitoring forexpression profile genes and/or corresponding polypeptides.

As described and defined herein, colorectal cancer proteins, includingintracellular, transmembrane or secreted proteins, find use as markersof colorectal cancer. Detection of these proteins in putative colorectalcancer tissue of patients allows for a determination or diagnosis ofcolorectal cancer. Numerous methods known to those of ordinary skill inthe art find use in detecting colorectal cancer. In one embodiment,antibodies are used to detect colorectal cancer proteins. A preferredmethod separates proteins from a sample or patient by electrophoresis ona gel (typically a denaturing and reducing protein gel, but may be anyother type of gel including isoelectric focusing gels and the like).Following separation of proteins, the colorectal cancer protein isdetected by immunoblotting with antibodies raised against the colorectalcancer protein. Methods of immunoblotting are well known to those ofordinary skill in the art.

In another preferred method, antibodies to the colorectal cancer proteinfind use in in situ imaging techniques. In this method cells arecontacted with from one to many antibodies to the colorectal cancerprotein(s). Following washing to remove non-specific antibody binding,the presence of the antibody or antibodies is detected. In oneembodiment the antibody is detected by incubating with a secondaryantibody that contains a detectable label. In another method the primaryantibody to the colorectal cancer protein(s) contains a detectablelabel. In another preferred embodiment each one of multiple primaryantibodies contains a distinct and detectable label. This method findsparticular use in simultaneous screening for a plurality of colorectalcancer proteins. As will be appreciated by one of ordinary skill in theart, numerous other histological imaging techniques are useful in theinvention.

In a preferred embodiment the label is detected in a fluorometer whichhas the ability to detect and distinguish emissions of differentwavelengths. In addition, a fluorescence activated cell sorter (FACS)can be used in the method.

In another preferred embodiment, antibodies find use in diagnosingcolorectal cancer from blood samples. As previously described, certaincolorectal cancer proteins are secreted/circulating molecules. Bloodsamples, therefore, are useful as samples to be probed or tested for thepresence of secreted colorectal cancer proteins. Antibodies can be usedto detect the colorectal cancer by any of the previously describedimmunoassay techniques including ELISA, immunoblotting (Westernblotting), immunoprecipitation, BIACORE technology and the like, as willbe appreciated by one of ordinary skill in the art.

In a preferred embodiment, in situ hybridization of labeled colorectalcancer nucleic acid probes to tissue arrays is done. For example, arraysof tissue samples, including colorectal cancer tissue and/or normaltissue, are made. In situ hybridization as is known in the art can thenbe done.

It is understood that when comparing the fingerprints between anindividual and a standard, the skilled artisan can make a diagnosis aswell as a prognosis. It is further understood that the genes whichindicate the diagnosis may differ from those which indicate theprognosis.

In a preferred embodiment, the colorectal cancer proteins, antibodies,nucleic acids, modified proteins and cells containing colorectal cancersequences are used in prognosis assays. As above, gene expressionprofiles can be generated that correlate to colorectal cancer severity,in terms of long term prognosis. Again, this may be done on either aprotein or gene level, with the use of genes being preferred. As above,the colorectal cancer probes are attached to biochips for the detectionand quantification of colorectal cancer sequences in a tissue orpatient. The assays proceed as outlined for diagnosis.

In a preferred embodiment, any of the three classes of proteins asdescribed herein are used in drug screening assays. The colorectalcancer proteins, antibodies, nucleic acids, modified proteins and cellscontaining colorectal cancer sequences are used in drug screening assaysor by evaluating the effect of drug candidates on a “gene expressionprofile” or expression profile of polypeptides. In a preferredembodiment, the expression profiles are used, preferably in conjunctionwith high throughput screening techniques to allow monitoring forexpression profile genes after treatment with a candidate agent,Zlokamik, et al., Science 279, 84-8 (1998), Heid, 1996 #69.

In a preferred embodiment, the colorectal cancer proteins, antibodies,nucleic acids, modified proteins and cells containing the native ormodified colorectal cancer proteins are used in screening assays. Thatis, the present invention provides novel methods for screening forcompositions which modulate the colorectal cancer phenotype. As above,this can be done on an individual gene level or by evaluating the effectof drug candidates on a “gene expression profile”. In a preferredembodiment, the expression profiles are used, preferably in conjunctionwith high throughput screening techniques to allow monitoring forexpression profile genes after treatment with a candidate agent, seeZlokamik, supra.

Having identified the colorectal cancer genes herein, a variety ofassays may be executed. In a preferred embodiment, assays may be run onan individual gene or protein level. That is, having identified aparticular gene as up regulated in colorectal cancer, candidatebioactive agents may be screened to modulate this gene's response;preferably to down regulate the gene, although in some circumstances toup regulate the gene. “Modulation” thus includes both an increase and adecrease in gene expression. The preferred amount of modulation willdepend on the original change of the gene expression in normal versustumor tissue, with changes of at least 10%, preferably 50%, morepreferably 100-300%, and in some embodiments 300-1000% or greater. Thus,if a gene exhibits a 4 fold increase in tumor compared to normal tissue,a decrease of about four fold is desired; a 10 fold decrease in tumorcompared to normal tissue gives a 10 fold increase in expression for acandidate agent is desired.

As will be appreciated by those in the art, this may be done byevaluation at either the gene or the protein level; that is, the amountof gene expression may be monitored using nucleic acid probes and thequantification of gene expression levels, or, alternatively, the geneproduct itself can be monitored, for example through the use ofantibodies to the colorectal cancer protein and standard immunoassays.

In a preferred embodiment, gene expression monitoring is done and anumber of genes, i.e. an expression profile, is monitoredsimultaneously, although multiple protein expression monitoring can bedone as well.

In this embodiment, the colorectal cancer nucleic acid probes areattached to biochips as outlined herein for the detection andquantification of colorectal cancer sequences in a particular cell. Theassays are further described below.

Generally, in a preferred embodiment, a candidate bioactive agent isadded to the cells prior to analysis. Moreover, screens are provided toidentify a candidate bioactive agent which modulates colorectal cancer,modulates colorectal cancer proteins, binds to a colorectal cancerprotein, or interferes between the binding of a colorectal cancerprotein and an antibody.

The term “candidate bioactive agent” or “drug candidate” or grammaticalequivalents as used herein describes any molecule, e.g., protein,oligopeptide, small organic molecule, polysaccharide, polynucleotide,etc., to be tested for bioactive agents that are capable of directly orindirectly altering the colorectal cancer phenotype or the expression ofa colorectal cancer sequence, including both nucleic acid sequences andprotein sequences. In preferred embodiments, the bioactive agentsmodulate the expression profiles, or expression profile nucleic acids orproteins provided herein. In a particularly preferred embodiment, thecandidate agent suppresses a colorectal cancer phenotype, for example toa normal colon tissue fingerprint. Similarly, the candidate agentpreferably suppresses a severe colorectal cancer phenotype. Generally aplurality of assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration or below the level ofdetection.

In one aspect, a candidate agent will neutralize the effect of a CRCprotein. By “neutralize” is meant that activity of a protein is eitherinhibited or counter acted against so as to have substantially no effecton a cell.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Preferred small molecules are less than 2000, or less than 1500 or lessthan 1000 or less than 500 D. Candidate agents comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Particularly preferred arepeptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, the candidate bioactive agents are proteins.By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations.

In a preferred embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eucaryotic proteins may be madefor screening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the candidate bioactive agents are peptidesof from about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation of nucleicacid binding domains, the creation of cysteines, for cross-linking,prolines for SH-3 domains, serines, threonines, tyrosines or histidinesfor phosphorylation sites, etc., or to purines, etc.

In a preferred embodiment, the candidate bioactive agents are nucleicacids, as defined above.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eucaryotic genomes may be used as is outlined above forproteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

After the candidate agent has been added and the cells allowed toincubate for some period of time, the sample containing the targetsequences to be analyzed is added to the biochip. If required, thetarget sequence is prepared using known techniques. For example, thesample may be treated to lyse the cells, using known lysis buffers,electroporation, etc., with purification and/or amplification such asPCR occurring as needed, as will be appreciated by those in the art. Forexample, an in vitro transcription with labels covalently attached tothe nucleosides is done. Generally, the nucleic acids are labeled withbiotin-FITC or PE, or with cy3 or cy5.

In a preferred embodiment, the target sequence is labeled with, forexample, a fluorescent, a chemiluminescent, a chemical, or a radioactivesignal, to provide a means of detecting the target sequence's specificbinding to a probe. The label also can be an enzyme, such as, alkalinephosphatase or horseradish peroxidase, which when provided with anappropriate substrate produces a product that can be detected.Alternatively, the label can be a labeled compound or small molecule,such as an enzyme inhibitor, that binds but is not catalyzed or alteredby the enzyme. The label also can be a moiety or compound, such as, anepitope tag or biotin which specifically binds to streptavidin. For theexample of biotin, the streptavidin is labeled as described above,thereby, providing a detectable signal for the bound target sequence. Asknown in the art, unbound labeled streptavidin is removed prior toanalysis.

As will be appreciated by those in the art, these assays can be directhybridization assays or can comprise “sandwich assays”, which includethe use of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670,5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118,5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporatedby reference. In this embodiment, in general, the target nucleic acid isprepared as outlined above, and then added to the biochip comprising aplurality of nucleic acid probes, under conditions that allow theformation of a hybridization complex.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions asoutlined above. The assays are generally run under stringency conditionswhich allows formation of the label probe hybridization complex only inthe presence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order, withpreferred embodiments outlined below. In addition, the reaction mayinclude a variety of other reagents may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target.

Once the assay is run, the data is analyzed to determine the expressionlevels, and changes in expression levels as between states, ofindividual genes, forming a gene expression profile.

The screens are done to identify drugs or bioactive agents that modulatethe colorectal cancer phenotype. Specifically, there are several typesof screens that can be run. A preferred embodiment is in the screeningof candidate agents that can induce or suppress a particular expressionprofile, thus preferably generating the associated phenotype. That is,candidate agents that can mimic or produce an expression profile incolorectal cancer similar to the expression profile of normal colontissue is expected to result in a suppression of the colorectal cancerphenotype. Thus, in this embodiment, mimicking an expression profile, orchanging one profile to another, is the goal.

In a preferred embodiment, as for the diagnosis and prognosisapplications, having identified the colorectal cancer genes important inany one state, screens can be run to alter the expression of the genesindividually. That is, screening for modulation of regulation ofexpression of a single gene can be done; that is, rather than try tomimic all or part of an expression profile, screening for regulation ofindividual genes can be done. Thus, for example, particularly in thecase of target genes whose presence or absence is unique between twostates, screening is done for modulators of the target gene expression.

In a preferred embodiment, screening is done to alter the biologicalfunction of the expression product of the colorectal cancer gene. Again,having identified the importance of a gene in a particular state,screening for agents that bind and/or modulate the biological activityof the gene product can be run as is more fully outlined below.

Thus, screening of candidate agents that modulate the colorectal cancerphenotype either at the gene expression level or the protein level canbe done.

In addition screens can be done for novel genes that are induced inresponse to a candidate agent. After identifying a candidate agent basedupon its ability to suppress a colorectal cancer expression patternleading to a normal expression pattern, or modulate a single colorectalcancer gene expression profile so as to mimic the expression of the genefrom normal tissue, a screen as described above can be performed toidentify genes that are specifically modulated in response to the agent.Comparing expression profiles between normal tissue and agent treatedcolorectal cancer tissue reveals genes that are not expressed in normalcolon tissue or colorectal cancer tissue, but are expressed in agenttreated tissue. These agent specific sequences can be identified andused by any of the methods described herein for colorectal cancer genesor proteins. In particular these sequences and the proteins they encodefind use in marking or identifying agent treated cells. In addition,antibodies can be raised against the agent induced proteins and used totarget novel therapeutics to the treated colorectal cancer tissuesample.

Thus, in one embodiment, a candidate agent is administered to apopulation of colorectal cancer cells, that thus has an associatedcolorectal cancer expression profile. By “administration” or“contacting” herein is meant that the candidate agent is added to thecells in such a manner as to allow the agent to act upon the cell,whether by uptake and intracellular action, or by action at the cellsurface. In some embodiments, nucleic acid encoding a proteinaceouscandidate agent (i.e. a peptide) may be put into a viral construct suchas a retroviral construct and added to the cell, such that expression ofthe peptide agent is accomplished; see PCT US97/01019, hereby expresslyincorporated by reference.

Once the candidate agent has been administered to the cells, the cellscan be washed if desired and are allowed to incubate under preferablyphysiological conditions for some period of time. The cells are thenharvested and a new gene expression profile is generated, as outlinedherein.

Thus, for example, colorectal cancer tissue may be screened for agentsthat reduce or suppress the colorectal cancer phenotype. A change in atleast one gene of the expression profile indicates that the agent has aneffect on colorectal cancer activity. By defining such a signature forthe colorectal cancer phenotype, screens for new drugs that alter thephenotype can be devised. With this approach, the drug target need notbe known and need not be represented in the original expressionscreening platform, nor does the level of transcript for the targetprotein need to change.

In a preferred embodiment, as outlined above, screens may be done onindividual genes and gene products (proteins). That is, havingidentified a particular colorectal cancer gene as important in aparticular state, screening of modulators of either the expression ofthe gene or the gene product itself can be done. The gene products ofcolorectal cancer genes are sometimes referred to herein as “colorectalcancer proteins” or “colorectal cancer modulating proteins” or “BCMP”.Additionally, “modulator” and “modulating” proteins are sometimes usedinterchangeably herein. In one embodiment, the colorectal cancer proteinis termed CJA8. CJA8 sequences can be identified as described herein forcolorectal cancer sequences. In one embodiment, CJA8 protein sequencesare as depicted in FIG. 2 (SEQ ID NO:2). The colorectal cancer proteinmay be a fragment, or alternatively, be the full length protein to thefragment shown herein. Preferably, the colorectal cancer protein is afragment. In a preferred embodiment, the amino acid sequence which isused to determine sequence identity or similarity is that depicted inFIG. 2. In another embodiment, the sequences are naturally occurringalelic variants of a protein having the sequence depicted in FIG. 2. Inanother embodiment, the sequences are sequence variants as furtherdescribed herein.

Preferably, the colorectal cancer protein is a fragment of approximately14 to 24 amino acids long. More preferably the fragment is a solublefragment. Preferably, the fragment includes a non-transmembrane region.In a preferred embodiment, the fragment has an N-terminal Cys to aid insolubility. In one embodiment, the c-terminus of the fragment is kept asa free acid and the n-terminus is a free amine to aid in coupling, i.e.,to cysteine. Preferably, the fragment of approximately 14 to 24 aminoacids long. More preferably the fragment is a soluble fragment. Inanother embodiment, a CJA8 fragment has at least one CJA8 bioactivity asdefined below.

In one embodiment the colorectal cancer proteins are conjugated to animmunogenic agent as discussed herein. In one embodiment the colorectalcancer protein is conjugated to BSA. In a preferred embodiment, asoutlined above, screens may be done on individual genes and geneproducts (proteins). That is, having identified a particular colorectalcancer gene as important in a particular state, screening of modulatorsof either the expression of the gene or the gene product itself can bedone. The gene products of colorectal cancer genes are sometimesreferred to herein as “colorectal cancer proteins” or “colorectal cancermodulating proteins” or “BCMP”. Additionally, “modulator” and“modulating” proteins are sometimes used interchangeably herein. In oneembodiment, the colorectal cancer protein is termed CJA8. CJA8 sequencescan be identified as described herein for colorectal cancer sequences.In one embodiment, CJA8 protein sequences are as depicted in FIG. 2 (SEQID NO:2). The colorectal cancer protein may be a fragment, oralternatively, be the full length protein to the fragment shown herein.Preferably, the colorectal cancer protein is a fragment. In a preferredembodiment, the amino acid sequence which is used to determine sequenceidentity or similarity is that depicted in FIG. 2. In anotherembodiment, the sequences are naturally occurring alelic variants of aprotein having the sequence depicted in FIG. 2. In another embodiment,the sequences are sequence variants as further described herein. Thus,in a preferred embodiment, screening for modulators of expression ofspecific genes can be done. This will be done as outlined above, but ingeneral the expression of only one or a few genes are evaluated.

In a preferred embodiment, screens are designed to first find candidateagents that can bind to colorectal cancer proteins, and then theseagents may be used in assays that evaluate the ability of the candidateagent to modulate colorectal cancer activity. Thus, as will beappreciated by those in the art, there are a number of different assayswhich may be run; binding assays and activity assays.

In a preferred embodiment, binding assays are done. In general, purifiedor isolated gene product is used; that is, the gene products of one ormore colorectal cancer nucleic acids are made. In general, this is doneas is known in the art. For example, antibodies are generated to theprotein gene products, and standard immunoassays are run to determinethe amount of protein present. Alternatively, cells comprising thecolorectal cancer proteins can be used in the assays.

Thus, in a preferred embodiment, the methods comprise combining acolorectal cancer protein and a candidate bioactive agent, anddetermining the binding of the candidate agent to the colorectal cancerprotein. Preferred embodiments utilize the human colorectal cancerprotein, although other mammalian proteins may also be used, for examplefor the development of animal models of human disease. In someembodiments, as outlined herein, variant or derivative colorectal cancerproteins may be used.

Generally, in a preferred embodiment of the methods herein, thecolorectal cancer protein or the candidate agent is non-diffusably boundto an insoluble support having isolated sample receiving areas (e.g. amicrotiter plate, an array, etc.). It is understood that alternatively,soluble assays known in the art may be performed. The insoluble supportsmay be made of any composition to which the compositions can be bound,is readily separated from soluble material, and is otherwise compatiblewith the overall method of screening. The surface of such supports maybe solid or porous and of any convenient shape. Examples of suitableinsoluble supports include microtiter plates, arrays, membranes andbeads. These are typically made of glass, plastic (e.g., polystyrene),polysaccharides, nylon or nitrocellulose, teflon™, etc. Microtiterplates and arrays are especially convenient because a large number ofassays can be carried out simultaneously, using small amounts ofreagents and samples. The particular manner of binding of thecomposition is not crucial so long as it is compatible with the reagentsand overall methods of the invention, maintains the activity of thecomposition and is nondiffusable. Preferred methods of binding includethe use of antibodies (which do not sterically block either the ligandbinding site or activation sequence when the protein is bound to thesupport), direct binding to “sticky” or ionic supports, chemicalcrosslinking, the synthesis of the protein or agent on the surface, etc.Following binding of the protein or agent, excess unbound material isremoved by washing. The sample receiving areas may then be blockedthrough incubation with bovine serum albumin (BSA), casein or otherinnocuous protein or other moiety.

In a preferred embodiment, the colorectal cancer protein is bound to thesupport, and a candidate bioactive agent is added to the assay.Alternatively, the candidate agent is bound to the support and thecolorectal cancer protein is added. Novel binding agents includespecific antibodies, non-natural binding agents identified in screens ofchemical libraries, peptide analogs, etc. Of particular interest arescreening assays for agents that have a low toxicity for human cells. Awide variety of assays may be used for this purpose, including labeledin vitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, functional assays(phosphorylation assays, etc.) and the like.

The determination of the binding of the candidate bioactive agent to thecolorectal cancer protein may be done in a number of ways. In apreferred embodiment, the candidate bioactive agent is labelled, andbinding determined directly. For example, this may be done by attachingall or a portion of the colorectal cancer protein to a solid support,adding a labelled candidate agent (for example a fluorescent label),washing off excess reagent, and determining whether the label is presenton the solid support. Various blocking and washing steps may be utilizedas is known in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal, e.g.radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the proteins (or proteinaceous candidate agents) may be labeled attyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, morethan one component may be labeled with different labels; using ¹²⁵I forthe proteins, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agentis determined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget molecule (i.e. colorectal cancer), such as an antibody, peptide,binding partner, ligand, etc. Under certain circumstances, there may becompetitive binding as between the bioactive agent and the bindingmoiety, with the binding moiety displacing the bioactive agent.

In one embodiment, the candidate bioactive agent is labeled. Either thecandidate bioactive agent, or the competitor, or both, is added first tothe protein for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high through put screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate bioactive agent. Displacement of the competitor is anindication that the candidate bioactive agent is binding to thecolorectal cancer protein and thus is capable of binding to, andpotentially modulating, the activity of the colorectal cancer protein.In this embodiment, either component can be labeled. Thus, for example,if the competitor is labeled, the presence of label in the wash solutionindicates displacement by the agent. Alternatively, if the candidatebioactive agent is labeled, the presence of the label on the supportindicates displacement.

In an alternative embodiment, the candidate bioactive agent is addedfirst, with incubation and washing, followed by the competitor. Theabsence of binding by the competitor may indicate that the bioactiveagent is bound to the colorectal cancer protein with a higher affinity.Thus, if the candidate bioactive agent is labeled, the presence of thelabel on the support, coupled with a lack of competitor binding, mayindicate that the candidate agent is capable of binding to thecolorectal cancer protein.

In a preferred embodiment, the methods comprise differential screeningto identity bioactive agents that are capable of modulating the activityof the colorectal cancer proteins. In this embodiment, the methodscomprise combining a colorectal cancer protein and a competitor in afirst sample. A second sample comprises a candidate bioactive agent, acolorectal cancer protein and a competitor. The binding of thecompetitor is determined for both samples, and a change, or differencein binding between the two samples indicates the presence of an agentcapable of binding to the colorectal cancer protein and potentiallymodulating its activity. That is, if the binding of the competitor isdifferent in the second sample relative to the first sample, the agentis capable of binding to the colorectal cancer protein.

Alternatively, a preferred embodiment utilizes differential screening toidentify drug candidates that bind to the native colorectal cancerprotein, but cannot bind to modified colorectal cancer proteins. Thestructure of the colorectal cancer protein may be modeled, and used inrational drug design to synthesize agents that interact with that site.Drug candidates that affect colorectal cancer bioactivity are alsoidentified by screening drugs for the ability to either enhance orreduce the activity of the protein.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

Screening for agents that modulate the activity of colorectal cancerproteins may also be done. In a preferred embodiment, methods forscreening for a bioactive agent capable of modulating the activity ofcolorectal cancer proteins comprise the steps of adding a candidatebioactive agent to a sample of colorectal cancer proteins, as above, anddetermining an alteration in the biological activity of colorectalcancer proteins. “Modulating the activity” of colorectal cancer includesan increase in activity, a decrease in activity, or a change in the typeor kind of activity present. Thus, in this embodiment, the candidateagent should both bind to colorectal cancer proteins (although this maynot be necessary), and alter its biological or biochemical activity asdefined herein. The methods include both in vitro screening methods, asare generally outlined above, and in vivo screening of cells foralterations in the presence, distribution, activity or amount ofcolorectal cancer proteins.

Thus, in this embodiment, the methods comprise combining a colorectalcancer sample and a candidate bioactive agent, and evaluating the effecton colorectal cancer activity. By “colorectal cancer activity” orgrammatical equivalents herein is meant at least one of colorectalcancer's biological activities, including, but not limited to, celldivision, preferably in colon tissue, cell proliferation, tumor growth,transformation of cells and serine protease activity. In one embodiment,colorectal cancer activity includes activation of CJA8 or a substratethereof by CJA8. An inhibitor of colorectal cancer activity is an agentwhich inhibits any one or more colorectal cancer activities.

In a preferred embodiment, the activity of the colorectal cancer proteinis increased; in another preferred embodiment, the activity of thecolorectal cancer protein is decreased. Thus, bioactive agents that areantagonists are preferred in some embodiments, and bioactive agents thatare agonists may be preferred in other embodiments.

In a preferred embodiment, the invention provides methods for screeningfor bioactive agents capable of modulating the activity of a colorectalcancer protein. The methods comprise adding a candidate bioactive agent,as defined above, to a cell comprising colorectal cancer proteins.Preferred cell types include almost any cell. The cells contain arecombinant nucleic acid that encodes a colorectal cancer protein. In apreferred embodiment, a library of candidate agents are tested on aplurality of cells.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, for examplehormones, antibodies, peptides, antigens, cytokines, growth factors,action potentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e. cell-cell contacts). Inanother example, the determinations are determined at different stagesof the cell cycle process.

In this way, bioactive agents are identified. Compounds withpharmacological activity are able to enhance or interfere with theactivity of the colorectal cancer protein. In one embodiment,“colorectal cancer protein activity” as used herein includes at leastone of the following: colorectal cancer activity, binding to CJA8,activation of CJA8 or activation of substrates of CJA8 by CJA8. Aninhibitor of CJA8 inhibits at least one of CJA8's bioactivities.

In one embodiment, a method of inhibiting colorectal cancer celldivision is provided. The method comprises administration of acolorectal cancer inhibitor.

In another embodiment, a method of inhibiting colorectal tumor growth isprovided. The method comprises administration of a colorectal cancerinhibitor. In a preferred embodiment, the inhibitor is an inhibitor ofCJA8.

In a further embodiment, methods of treating cells or individuals withcolorectal cancer are provided. The method comprises administration of acolorectal cancer inhibitor. In a preferred embodiment, the inhibitor isan inhibitor of CJA8.

In one embodiment, a colorectal cancer inhibitor is an antibody asdiscussed above. In another embodiment, the colorectal cancer inhibitoris an antisense molecule. Antisense molecules as used herein includeantisense or sense oligonucleotides comprising a singe-stranded nucleicacid sequence (either RNA or DNA) capable of binding to target mRNA(sense) or DNA (antisense) sequences for colorectal cancer molecules. Apreferred antisense molecule is for CJA8 or for a ligand or activatorthereof. Antisense or sense oligonucleotides, according to the presentinvention, comprise a fragment generally at least about 14 nucleotides,preferably from about 14 to 30 nucleotides. The ability to derive anantisense or a sense oligonucleotide, based upon a cDNA sequenceencoding a given protein is described in, for example, Stein and Cohen(Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques6:958, 1988).

Antisense molecules may be introduced into a cell containing the targetnucleotide sequence by formation of a conjugate with a ligand bindingmolecule, as described in WO 91/04753. Suitable ligand binding moleculesinclude, but are not limited to, cell surface receptors, growth factors,other cytokines, or other ligands that bind to cell surface receptors.Preferably, conjugation of the ligand binding molecule does notsubstantially interfere with the ability of the ligand binding moleculeto bind to its corresponding molecule or receptor, or block entry of thesense or antisense oligonucleotide or its conjugated version into thecell. Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. It is understood that the use of antisense molecules or knockout and knock in models may also be used in screening assays asdiscussed above, in addition to methods of treatment.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host, aspreviously described. The agents may be administered in a variety ofways, orally, parenterally e.g., subcutaneously, intraperitoneally,intravascularly, etc. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways. The concentration oftherapeutically active compound in the formulation may vary from about0.1-100 wt. %. The agents may be administered alone or in combinationwith other treatments, i.e., radiation.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

Without being bound by theory, it appears that the various colorectalcancer sequences are important in colorectal cancer. Accordingly,disorders based on mutant or variant colorectal cancer genes may bedetermined. In one embodiment, the invention provides methods foridentifying cells containing variant colorectal cancer genes comprisingdetermining all or part of the sequence of at least one endogeneouscolorectal cancer gene in a cell. As will be appreciated by those in theart, this may be done using any number of sequencing techniques. In apreferred embodiment, the invention provides methods of identifying thecolorectal cancer genotype of an individual comprising determining allor part of the sequence of at least one colorectal cancer gene of theindividual. This is generally done in at least one tissue of theindividual, and may include the evaluation of a number of tissues ordifferent samples of the same tissue. The method may include comparingthe sequence of the sequenced gene to a known gene, i.e. a wild-typegene.

The sequence of all or part of the colorectal cancer gene can then becompared to the sequence of a known colorectal cancer gene to determineif any differences exist. This can be done using any number of knownhomology programs, such as Bestfit, etc. In a preferred embodiment, thepresence of a difference in the sequence between the colorectal cancergene of the patient and the known colorectal cancer gene is indicativeof a disease state or a propensity for a disease state, as outlinedherein.

In a preferred embodiment, the colorectal cancer genes are used asprobes to determine the number of copies of the colorectal cancer genein the genome.

In another preferred embodiment colorectal cancer genes are used asprobed to determine the chromosomal localization of the colorectalcancer genes. Information such as chromosomal localization finds use inproviding a diagnosis or prognosis in particular when chromosomalabnormalities such as translocations, and the like are identified incolorectal cancer gene loci.

Thus, in one embodiment, methods of modulating colorectal cancer incells or organisms are provided. In one embodiment, the methods compriseadministering to a cell an antibody that reduces or eliminates thebiological activity of an endogenous colorectal cancer protein.Alternatively, the methods comprise administering to a cell or organisma recombinant nucleic acid encoding a colorectal cancer protein. As willbe appreciated by those in the art, this may be accomplished in anynumber of ways. In a preferred embodiment, for example when thecolorectal cancer sequence is down-regulated in colorectal cancer, theactivity of the colorectal cancer gene is in creased by increasing theamount in the cell, for example by overexpressing the endogenous proteinor by administering a gene encoding the sequence, using knowngene-therapy techniques, for example. In a preferred embodiment, thegene therapy techniques include the incorporation of the exogenous geneusing enhanced homologous recombination (EHR), for example as describedin PCT/US93/03868, hereby incorporated by reference in its entirety.Alternatively, for example when the colorectal cancer sequence isup-regulated in colorectal cancer, the activity of the endogeneous geneis decreased, for example by the administration of an inhibitor ofcolorectal cancer, such as an antisense nucleic acid.

In one embodiment, the colorectal cancer proteins of the presentinvention may be used to generate polyclonal and monoclonal antibodiesto colorectal cancer proteins, which are useful as described herein.Similarly, the colorectal cancer proteins can be coupled, using standardtechnology, to affinity chromatography columns. These columns may thenbe used to purify colorectal cancer antibodies. In a preferredembodiment, the antibodies are generated to epitopes unique to acolorectal cancer protein; that is, the antibodies show little or nocross-reactivity to other proteins. These antibodies find use in anumber of applications. For example, the colorectal cancer antibodiesmay be coupled to standard affinity chromatography columns and used topurify colorectal cancer proteins. The antibodies may also be used asblocking polypeptides, as outlined above, since they will specificallybind to the colorectal cancer protein.

In one embodiment, a therapeutically effective dose of a colorectalcancer or modulator thereof is administered to a patient. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. As is known in the art, adjustmentsfor degradation, systemic versus localized delivery, and rate of newprotease synthesis, as well as the age, body weight, general health,sex, diet, time of administration, drug interaction and the severity ofthe condition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

The administration of the colorectal cancer proteins and modulators ofthe present invention can be done in a variety of ways as discussedabove, including, but not limited to, orally, subcutaneously,intravenously, intranasally, transdermally, intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.In some instances, for example, in the treatment of wounds andinflammation, the colorectal cancer proteins and modulators may bedirectly applied as a solution or spray.

The pharmaceutical compositions of the present invention comprise acolorectal cancer protein in a form suitable for administration to apatient. In the preferred embodiment, the pharmaceutical compositionsare in a water soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

In a preferred embodiment, colorectal cancer proteins and modulators areadministered as therapeutic agents, and can be formulated as outlinedabove. Similarly, colorectal cancer genes (including both thefull-length sequence, partial sequences, or regulatory sequences of thecolorectal cancer coding regions) can be administered in gene therapyapplications, as is known in the art. These colorectal cancer genes caninclude antisense applications, either as gene therapy (i.e. forincorporation into the genome) or as antisense compositions, as will beappreciated by those in the art.

In a preferred embodiment, colorectal cancer genes are administered asDNA vaccines, either single genes or combinations of colorectal cancergenes. Naked DNA vaccines are generally known in the art. Brower, NatureBiotechnology, 16:1304-1305 (1998).

In one embodiment, colorectal cancer genes of the present invention areused as DNA vaccines. Methods for the use of genes as DNA vaccines arewell known to one of ordinary skill in the art, and include placing acolorectal cancer gene or portion of a colorectal cancer gene under thecontrol of a promoter for expression in a patient with colorectalcancer. The colorectal cancer gene used for DNA vaccines can encodefull-length colorectal cancer proteins, but more preferably encodesportions of the colorectal cancer proteins including peptides derivedfrom the colorectal cancer protein. In a preferred embodiment a patientis immunized with a DNA vaccine comprising a plurality of nucleotidesequences derived from a colorectal cancer gene. Similarly, it ispossible to immunize a patient with a plurality of colorectal cancergenes or portions thereof as defined herein. Without being bound bytheory, expression of the polypeptide encoded by the DNA vaccine,cytotoxic T-cells, helper T-cells and antibodies are induced whichrecognize and destroy or eliminate cells expressing colorectal cancerproteins.

In a preferred embodiment, the DNA vaccines include a gene encoding anadjuvant molecule with the DNA vaccine. Such adjuvant molecules includecytokines that increase the immunogenic response to the colorectalcancer polypeptide encoded by the DNA vaccine. Additional or alternativeadjuvants are known to those of ordinary skill in the art and find usein the invention.

In another preferred embodiment colorectal cancer genes find use ingenerating animal models of colorectal cancer. For example, as isappreciated by one of ordinary skill in the art, when the colorectalcancer gene identified is repressed or diminished in colorectal cancertissue, gene therapy technology wherein antisense RNA directed to thecolorectal cancer gene will also diminish or repress expression of thegene. An animal generated as such serves as an animal model ofcolorectal cancer that finds use in screening bioactive drug candidates.Similarly, gene knockout technology, for example as a result ofhomologous recombination with an appropriate gene targeting vector, willresult in the absence of the colorectal cancer protein. When desired,tissue-specific expression or knockout of the colorectal cancer proteinmay be necessary.

It is also possible that the colorectal cancer protein is overexpressedin colorectal cancer. As such, transgenic animals can be generated thatoverexpress the colorectal cancer protein. Depending on the desiredexpression level, promoters of various strengths can be employed toexpress the transgene. Also, the number of copies of the integratedtransgene can be determined and compared for a determination of theexpression level of the transgene. Animals generated by such methodsfind use as animal models of colorectal cancer and are additionallyuseful in screening for bioactive molecules to treat disorders relatedto the colorectal cancer protein.

It is understood that the examples described herein in no way serve tolimit the true scope of this invention, but rather are presented forillustrative purposes. All references and sequences of accession numberscited herein are incorporated by reference in their entirety.

EXAMPLES Example 1 Tissue Preparation, Labeling Chips, and Fingerprints

Purify Total RNA from Tissue Using TRIzol Reagent

Estimate tissue weight. Homogenize tissue samples in 1 ml of TRIzol per50 mg of tissue using a Polytron 3100 homogenizer. The generator/probeused depends upon the tissue size. A generator that is too large for theamount of tissue to be homogenized will cause a loss of sample and lowerRNA yield. Use the 20 mm generator for tissue weighing more than 0.6 g.If the working volume is greater than 2 ml, then homogenize tissue in a15 ml polypropylene tube (Falcon 2059). Fill tube no greater than 10 ml.

Homogenization

Before using generator, it should have been cleaned after last usage byrunning it through soapy H₂O and rinsing thoroughly. Run through withEtOH to sterilize. Keep tissue frozen until ready. Add TRIzol directlyto frozen tissue then homogenize.

Following homogenization, remove insoluble material from the homogenateby centrifugation at 7500×g for 15 min. in a Sorvall superspeed or12,000×g for 10 min. in an Eppendorf centrifuge at 4° C. Transfer thecleared homogenate to a new tube(s). The samples may be frozen now at−60 to −70° C. (and kept for at least one month) or you may continuewith the purification.

Phase Separation

Incubate the homogenized samples for 5 minutes at room temperature. Add0.2 ml of chloroform per 1 ml of TRIzol reagent used in the originalhomogenization. Cap tubes securely and shake tubes vigorously by hand(do not vortex) for 15 seconds. Incubate samples at room temp. for 2-3minutes. Centrifuge samples at 6500 rpm in a Sorvall superspeed for 30min. at 4° C. (You may spin at up to 12,000×g for 10 min. but you riskbreaking your tubes in the centrifuge.)

RNA Precipitation

Transfer the aqueous phase to a fresh tube. Save the organic phase ifisolation of DNA or protein is desired. Add 0.5 ml of isopropyl alcoholper 1 ml of TRizol reagent used in the original homogenization. Captubes securely and invert to mix. Incubate samples at room temp. for 10minutes. Centrifuge samples at 6500 rpm in Sorvall for 20 min. at 4° C.

RNA Wash

Pour off the supernate. Wash pellet with cold 75% ethanol. Use 1 ml of75% ethanol per 1 ml of TRIzol reagent used in the initialhomogenization. Cap tubes securely and invert several times to loosenpellet. (Do not vortex). Centrifuge at <800 rpm (<7500×g) for 5 minutesat 4° C. Pour off the wash. Carefully transfer pellet to an eppendorftube (let it slide down the tube into the new tube and use a pipet tipto help guide it in if necessary). Depending on the volumes you areworking with, you can decide what size tube(s) you want to precipitatethe RNA in. When I tried leaving the RNA in the large 15 ml tube, ittook so long to dry (i.e. it did not dry) that I eventually had totransfer it to a smaller tube. Let pellet dry in hood. Resuspend RNA inan appropriate volume of DEPC H₂O. Try for 2-5 ug/ul. Take absorbancereadings.

Purify Poly A+ mRNA from Total RNA or Clean Up Total RNA with Qiagen'sRNeasy Kit

Purification of poly A⁺ mRNA from total RNA. Heat oligotex suspension to37° C. and mix immediately before adding to RNA. Incubate Elution Bufferat 70° C. Warm up 2×Binding Buffer at 65° C. if there is precipitate inthe buffer. Mix total RNA with DEPC-treated water, 2×Binding Buffer, andOligotex according to Table 2 on page 16 of the Oligotex Handbook.Incubate for 3 minutes at 65° C. Incubate for 10 minutes at roomtemperature. Centrifuge for 2 minutes at 14,000 to 18,000 g. Ifcentrifuge has a “soft setting,” then use it. Remove supernatant withoutdisturbing Oligotex pellet. A little bit of solution can be left behindto reduce the loss of Oligotex. Save sup until certain that satisfactorybinding and elution of poly A mRNA has occurred.

Gently resuspend in Wash Buffer OW2 and pipet onto spin column.Centrifuge the spin column at full speed (soft setting if possible) for1 minute.

Transfer spin column to a new collection tube and gently resuspend inWash Buffer OW2 and centrifuge as describe herein.

Transfer spin column to a new tube and elute with 20 to 100 ul ofpreheated (70° C.) Elution Buffer. Gently resuspend Oligotex resin bypipetting up and down. Centrifuge as above. Repeat elution with freshelution buffer or use first eluate to keep the elution volume low.

Read absorbance, using diluted Elution Buffer as the blank.

Before proceeding with cDNA synthesis, the mRNA must be precipitated.Some component leftover or in the Elution Buffer from the Oligotexpurification procedure will inhibit downstream enzymatic reactions ofthe mRNA.

Ethanol Precipitation

Add 0.4 vol. of 7.5 M NH₄OAc+2.5 vol. of cold 100% ethanol. Precipitateat −20° C. 1 hour to overnight (or 20-30 min. at −70° C.). Centrifuge at14,000-16,000×g for 30 minutes at 4 pellet with 0.5 ml of 80%ethanol(−20° C.) then centrifuge at 14,000-16,000×g for 5 minutes at roomtemperature. Repeat 80% ethanol wash. Dry the last bit of ethanol fromthe pellet in the hood. (Do not speed vacuum). Suspend pellet in DEPCH₂0 at 1 μg/ul concentration.

Clean Up Total RNA Using Qiagen's RNeasy Kit

Add no more than 10 ug to an RNeasy column. Adjust sample to a volume of100 ul with RNase-free water. Add 350 ul Buffer RLT then 250 ul ethanol(100%) to the sample. Mix by pipetting (do not centrifuge) then applysample to an RNeasy mini spin column. Centrifuge for 15 sec at >10,000rpm. If concerned about yield, re-apply flowthrough to column andcentrifuge again.

Transfer column to a new 2-ml collection tube. Add 500 ul Buffer RPE andcentrifuge for 15 sec at >10,000 rpm. Discard flowthrough. Add 500 ulBuffer RPE and centrifuge for 15 sec at >10,000 rpm. Discard flowthroughthen centrifuge for 2 min at maximum speed to dry column membrane.Transfer column to a new 1.5-ml collection tube and apply 30-50 ul ofRNase-free water directly onto column membrane. Centrifuge 1 minat >10,000 rpm. Repeat elution. Take absorbance reading. If necessary,ethanol precipitate with ammonium acetate and 2.5×volume 100% ethanol.

Make cDNA Using Gibco's “SuperScript Choice System for cDNA Synthesis”Kit First Strand cDNA Synthesis

Use 5 ug of total RNA or 1 ug of polyA+ mRNA as starting material. Fortotal RNA, use 2 ul of SuperScript RT. For polyA+ mRNA, use 1 ul ofSuperScript RT. Final volume of first strand synthesis mix is 20 ul. RNAmust be in a volume no greater than 10 ul. Incubate RNA with 1 ul of 100pmol T7-T24 oligo for 10 min at 70 C. On ice, add 7 ul of: 4 ul 5×1^(st)Strand Buffer, 2 ul of 0.1M DTT, and 1 ul of 10 mM dNTP mix. Incubate at37 C. for2 min then add SuperScript RT Incubate at 37 C. for 1 hour.

Second Strand Synthesis

Place 1^(st) strand reactions on ice.

Add:

91 ul DEPC H20

30 ul 5×2^(nd) Strand Buffer

3 ul 10 mM dNTP mix

1 ul 10 U/ul E.coli DNA Ligase

4 ul 10 U/ul E.coli DNA Polymerase

1 ul 2 U/ul RNase H

Make the above into a mix if there are more than 2 samples. Mix andincubate 2 hours at 16 C. Add 2 ul T4 DNA Polymerase. Incubate 5 min at16 C. Add 10 ul of 0.5M EDTA

Clean Up cDNA

Phenol:Chloroform:Isoamyl Alcohol (25:24:1) purification usingPhase-Lock gel tubes: Centrifuge PLG tubes for 30 sec at maximum speed.Transfer cDNA mix to PLG tube. Add equal volume ofphenol:chloroform:isamyl alcohol and shake vigorously (do not vortex).Centrifuge 5 minutes at maximum speed. Transfer top aqueous solution toa new tube. Ethanol precipitate: add 7.5×5M NH4Oac and 2.5×volume of100% ethanol. Centrifuge immediately at room temp. for 20 min, maximumspeed. Remove sup then wash pellet 2× with cold 80% ethanol. Remove asmuch ethanol wash as possible then let pellet air dry. Resuspend pelletin 3 ul RNase-free water.

In vitro Transcription (IVT) and Labeling with Biotin

Pipet 1.5 ul of cDNA into a thin-wall PCR tube.

Make NTP Labeling Mix

Combine at room 2 ul T7 10xATP (75 mM) (Ambion) temperature: 2 ul T710xGTP (75 mM) (Ambion) 1.5 ul T7 10xCTP (75 mM) (Ambion) 1.5 ul T710xUTP (75 mM) (Ambion) 3.75 ul 10 mM Bio-11-UTP (Boehringer-Mannheim/Roche or Enzo) 3.75 ul 10 mM Bio-16-CTP (Enzo) 2 ul 10x T7transcription buffer (Ambion) 2 ul 10x T7 enzyme mix (Ambion)

Final volume of total reaction is 20 ul. Incubate 6 hours at 37° C. in aPCR machine.

RNeasy Clean-up of IVT Product

Follow previous instructions for RNeasy columns or refer to Qiagen'sRNeasy protocol handbook.

cRNA will most likely need to be ethanol precipitated. Resuspend in avolume compatible with the fragmentation step.

Fragmentation

15 ug of labeled RNA is usually fragmented. Try to minimize thefragmentation reaction volume; a 10 ul volume is recommended but 20 ulis all right. Do not go higher than 20 ul because the magnesium in thefragmentation buffer contributes to precipitation in the hybridizationbuffer. Fragment RNA by incubation at 94 C for 35 minutes in1×Fragmentation buffer.

5×Fragmentation Buffer

200 mM Tris-acetate, pH 8.1

500 mM KOAc

150 mM MgOAc

The labeled RNA transcript can be analyzed before and afterfragmentation. Samples can be heated to 65 C. for 15 minutes andelectrophoresed on 1% agarose/TBE gels to get an approximate idea of thetranscript size range.

Hybridization

200 ul (10 ug cRNA) of a hybridization mix is put on the chip. Ifmultiple hybridizations are to be done (such as cycling through a 5 chipset), then it is recommended that an initial hybridization mix of 300 ulor more be made.

Hybrization Mix: Fragment Labeled RNA (50 ng/ul Final Conc.)

50 pM 948-b control oligo

1.5 pM BioB

5 pM BioC

25 pM BioD

100 pM CRE

0.1 mg/ml herring sperm DNA

0.5 mg/ml acetylated BSA

to 300 ul with 1×MES hyb. buffer

The instruction manuals for the products used herein are incorporatedherein in their entirety.

Labeling Protocol Provided Herein Hybridization reaction: Start withnon-biotinylated IVT (purified by RNeasy columns) (see example 1 forsteps from tissue to IVT) IVT antisense RNA; 4 μg: μl Random Hexamers (1μg/μl): 4 μl H₂O: μl 14 μl

Incubate 70° C., 10 min. Put on ice.

Reverse Transcription

5X First Strand (BRL) buffer: 6 μl 0.1 M DTT: 3 μl 50X dNTP mix: 0.6 μlH₂O: 2.4 μl Cy3 or Cy5 dUTP (1 mM): 3 μl SS RT II (BRL): 1 μl 16 μl

Add to hybridization reaction.

Incubate 30 min., 42° C.

Add 1 μl SSII and let go for another hour.

Put on ice.

50×dNTP mix (25 mM of cold dATP, dCTP, and dGTP, 10 mM of dTTP: 25 μieach of 100 mM dATP, dCTP, and dGTP; 10 μl of 100 mM dTTP to 15 μl H2O.dNTPs from Pharmacia)

RNA Degradation

- Add 1.5 μl 1M NaOH/2 mM EDTA, 86 μl H₂O incubate at 65° C., 10 min. 10μl 10N NaOH  4 μl 50 mM EDTA

U-Con 30

500 μl TE/sample spin at 7000 g for 10 min, save flow through forpurification

Qiagen Purification

suspend u-con recovered material in 500 μl buffer PB

proceed w/ normal Qiagen protocol

DNAse Digest

Add 1 μl of 1/100 dil of DNAse/30 μl Rx and incubate at 37° C. for 15min.

5 min 95° C. to denature enzyme

Sample Preparation

-Add: Cot-1 DNA: 10 μl 50X dNTPs: 1 μl 20X SSC: 2.3 μl Na pyrophosphate: 7.5 μl 10 mg/ml Herring sperm DNA 1 μl of 1/10 dilution 21.8final vol.

Dry down in speed vac.

Resuspend in 15 μl H₂O.

Add 0.38 μl 10% SDS.

Heat 95° C., 2 min.

Slow cool at room temp. for 20 min.

Put on slide and hybridize overnight at 64° C.

Washing After the Hybridization

3X SSC/0.03% SDS: 2 min. 37.5 mls 20X SSC + 0.75 mls 10% SDS in 250 mlsH₂O 1X SSC: 5 min. 12.5 mls 20X SSC in 250 mls H₂O 0.2X SSC: 5 min. 2.5mls 20X SSC in 250 mls H₂O

Dry slides in centrifuge, 1000 RPM, 1 min.

Scan at appropriate PMT's and channels.

Example 2

Expression studies were performed herein. As indicated in FIG. 3, CJA8is upregulated in colorectal cancer tissue. CJA8 is located onchromosome 11.

3 1 2079 DNA Homo sapiens 1 gagaggcagc agcttgttca gcggacaagg atgctgggcgtgagggacca aggcctgccc 60 tgcactcggg cctcctccag ccagtgctga ccagggacttctgacctgct ggccagccag 120 gacctgtgtg gggaggccct cctgctgcct tggggtgacaatctcagctc caggctacag 180 ggagaccggg aggatcacag agccagcatg gtacaggatcctgacagtga tcaacctctg 240 aacagcctcg atgtcaaacc cctgcgcaaa ccccgtatccccatggagac cttcagaaag 300 tgtggggatc cccatcatca tagcactact gagcctggcgagtatcatca ttgtggttgt 360 cctcatcaag gtgattctgg ataaatacta cttcctctgcgggcagcctc tccacttcat 420 cccgaggaag cagctgtgtg acggagagct ggactgtcccttgggggagg acgaggagca 480 ctgtgtcaag agcttccccg aagggcctgc agtggcagtccgcctctcca aggaccgatc 540 cacactgcag gtgctggact cggccacagg gaactggttctctgcctgtt tcgacaactt 600 cacagaagct ctcgctgaga cagcctgtag gcagatgggctacagcagca aacccacttt 660 cagagctgtg gagattggcc cagaccagga tctggatgttgttgaaatca cagaaaacag 720 ccaggagctt cgcatgcgga actcaagtgg gccctgtctctcaggctccc tggtctccct 780 gcactgtctt gcctgtggga agagcctgaa gaccccccgtgtggtgggtg gggaggaggc 840 ctctgtggat tcttggcctt ggcaggtcag catccagtacgacaaacagc acgtctgtgg 900 agggagcatc ctggaccccc actgggtcct cacggcagcccactgcttca ggaaacatac 960 cgatgtgttc aactggaagg tgcgggcagg ctcagacaaactgggcagct tcccatccct 1020 ggctgtggcc aagatcatca tcattgaatt caaccccatgtaccccaaag acaatgacat 1080 cgccctcatg aagctgcagt tcccactcac tttctcaggcacagtcaggc ccatctgtct 1140 gcccttcttt gatgaggagc tcactccagc caccccactctggatcattg gatggggctt 1200 tacgaagcag aatggaggga agatgtctga catactgctgcaggcgtcag tccaggtcat 1260 tgacagcaca cggtgcaatg cagacgatgc gtaccagggggaagtcaccg agaagatgat 1320 gtgtgcaggc atcccggaag ggggtgtgga cacctgccagggtgacagtg gtgggcccct 1380 gatgtaccaa tctgaccagt ggcatgtggt gggcatcgttagctggggct atggctgcgg 1440 gggcccgagc accccaggag tatacaccaa ggtctcagcctatctcaact ggatctacaa 1500 tgtctggaag gctgagctgt aatgctgctg cccctttgcagtgctgggag ccgcttcctt 1560 cctgccctgc ccacctgggg atcccccaaa gtcagacacagagcaagagt ccccttgggt 1620 acacccctct gcccacagcc tcagcatttc ttggagcagcaaagggcctc aattcctgta 1680 agagaccctc gcagcccaga ggcgcccaga ggaagtcagcagccctagct cggccacact 1740 tggtgctccc agcatcccag ggagagacac agcccactgaacaaggtctc aggggtattg 1800 ctaagccaag aaggaacttt cccacactac tgaatggaagcaggctgtct tgtaaaagcc 1860 cagatcactg tgggctggag aggagaagga aagggtctgcgccagccctg tccgtcttca 1920 cccatcccca agcctactag agcaagaaac cagttgtaatataaaatgca ctgccctact 1980 gttggtatga ctaccgttac ctactgttgt cattgttattacagctatgg ccactattat 2040 taaagagctg tgtaacatca aaaaaaaaaa aaaaaaaaa2079 2 423 PRT Homo sapiens 2 Met Ser Asn Pro Cys Ala Asn Pro Val SerPro Trp Arg Pro Ser Glu 1 5 10 15 Ser Val Gly Ile Pro Leu Ile Ile AlaLeu Leu Ser Leu Ala Ser Ile 20 25 30 Ile Ile Val Val Val Leu Ile Lys ValIle Leu Asp Lys Tyr Tyr Phe 35 40 45 Leu Cys Gly Gln Pro Leu His Phe IlePro Arg Lys Gln Leu Cys Asp 50 55 60 Gly Glu Leu Asp Cys Pro Leu Gly GluAsp Glu Glu His Cys Val Lys 65 70 75 80 Ser Phe Pro Glu Gly Pro Ala ValAla Val Arg Leu Ser Lys Asp Arg 85 90 95 Ser Thr Leu Gln Val Leu Asp SerAla Thr Gly Asn Trp Phe Ser Ala 100 105 110 Cys Phe Asp Asn Phe Thr GluAla Leu Ala Glu Thr Ala Cys Arg Gln 115 120 125 Met Gly Tyr Ser Ser LysPro Thr Phe Arg Ala Val Glu Ile Gly Pro 130 135 140 Asp Gln Asp Leu AspVal Val Glu Ile Thr Glu Asn Ser Gln Glu Leu 145 150 155 160 Arg Met ArgAsn Ser Ser Gly Pro Cys Leu Ser Gly Ser Leu Val Ser 165 170 175 Leu HisCys Leu Ala Cys Gly Lys Ser Leu Lys Thr Pro Arg Val Val 180 185 190 GlyGly Glu Glu Ala Ser Val Asp Ser Trp Pro Trp Gln Val Ser Ile 195 200 205Gln Tyr Asp Lys Gln His Val Cys Gly Gly Ser Ile Leu Asp Pro His 210 215220 Trp Val Leu Thr Ala Ala His Cys Phe Arg Lys His Thr Asp Val Phe 225230 235 240 Asn Trp Lys Val Arg Ala Gly Ser Asp Lys Leu Gly Ser Phe ProSer 245 250 255 Leu Ala Val Ala Lys Ile Ile Ile Ile Glu Phe Asn Pro MetTyr Pro 260 265 270 Lys Asp Asn Asp Ile Ala Leu Met Lys Leu Gln Phe ProLeu Thr Phe 275 280 285 Ser Gly Thr Val Arg Pro Ile Cys Leu Pro Phe PheAsp Glu Glu Leu 290 295 300 Thr Pro Ala Thr Pro Leu Trp Ile Ile Gly TrpGly Phe Thr Lys Gln 305 310 315 320 Asn Gly Gly Lys Met Ser Asp Ile LeuLeu Gln Ala Ser Val Gln Val 325 330 335 Ile Asp Ser Thr Arg Cys Asn AlaAsp Asp Ala Tyr Gln Gly Glu Val 340 345 350 Thr Glu Lys Met Met Cys AlaGly Ile Pro Glu Gly Gly Val Asp Thr 355 360 365 Cys Gln Gly Asp Ser GlyGly Pro Leu Met Tyr Gln Ser Asp Gln Trp 370 375 380 His Val Val Gly IleVal Ser Trp Gly Tyr Gly Cys Gly Gly Pro Ser 385 390 395 400 Thr Pro GlyVal Tyr Thr Lys Val Ser Ala Tyr Leu Asn Trp Ile Tyr 405 410 415 Asn ValTrp Lys Ala Glu Leu 420 3 5 PRT Unknown cytokine receptor extracellularmotif found in many species 3 Trp Ser Xaa Trp Ser 1 5

We claim:
 1. A method of diagnosing colorectal cancer comprising: a)determining the expression of a gene encoding CJA8 or a fragment thereofin a first colon tissue of a first individual; and b) comparing saidexpression of said gene(s) to the expression of a gene encoding CJA8 ora fragment thereof in a second normal tissue from said first individualor in a tissue from a second unaffected individual; wherein a differencein said expression indicates that the first individual has colorectalcancer.
 2. The method of claim 1, wherein said second normal tissue ofsaid first individual is colorectal tissue.
 3. The method of claim 2,wherein a difference between expression in said first colon tissue andsaid second normal tissue indicates that the first individual hascolorectal cancer.
 4. The method of claim 1, wherein said second normaltissue of said first individual is not colorectal tissue.
 5. The methodof claim 4, wherein a difference between expression in said first colontissue and said second normal tissue indicates that the first individualhas colorectal cancer.
 6. The method of claim 1, wherein said tissue ofsaid second unaffected individual is normal colorectal tissue.
 7. Themethod of claim 6, wherein a difference between expression in said firstcolon tissue and said tissue of said second unaffected individualindicates that the first individual has colorectal cancer.
 8. The methodof claim 1, wherein said tissue of said second unaffected individual iscolorectal cancer tissue.
 9. The method of claim 8, wherein a similaritybetween expression in said first colon tissue and said tissue of saidsecond unaffected individual indicates that the first individual hascolorectal cancer.
 10. The method of claim 1, wherein said determiningis by measuring RNA encoding CJA8.
 11. The method of claim 10, whereinsaid measuring utilizes a biochip comprising a nucleic acid comprisingthe sequence of FIG. 1 (SEQ ID NO:1) or a fragment thereof.
 12. Themethod of claim 10, wherein said RNA encoding CJA8 comprises the RNAequivalent of the sequence as disclosed in FIG. 1 (SEQ ID NO:1) or afragment thereof.
 13. The method of claim 1, wherein said determining isby measuring CJA8 protein.
 14. The method of claim 13, wherein saidmeasuring CJA8 protein comprises binding of CJA8 with an antibody. 15.The method of claim 14, wherein said antibody specifically binds apolypeptide comprising the sequence as disclosed in FIG. 2 (SEQ ID NO:2)or a fragment thereof.
 16. A method of diagnosing colorectal cancercomprising: a) determining the expression of a gene encoding CJA8 in afirst colon tissue of a first individual; and b) comparing saidexpression of said gene(s) to the expression of a gene encoding CJA8 ina second normal tissue from said first individual or in a tissue from asecond unaffected individual; wherein a difference in said expressionindicates that the first individual has colorectal cancer.